Silane-treated silica filter media

ABSTRACT

The present invention provides methods for separating one or more components of interest from a sample containing particulates and soluble materials. The method comprises the steps of: (a) filtering a sample through silica filter media whose surface silanol groups have reacted with one or more silanes, and (b) simultaneously capturing particulates and binding a soluble component to the silica filter media. The bound soluble component of interest is subsequently eluted from the silica filter media. In one embodiment of the invention, unwanted soluble materials are captured by the treated silica filter media and desired component of interest is recovered from the flow-through. In another embodiment, different components of interest are recovered from both the eluate and the flow-through. Preferred treated silica filter media are silane-treated rice hull ash or diatomaceous earth with functional quarternary ammonium group or functional sulphonate group. The present invention also provides silane-treated silica filter media.

This application is a continuation of U.S. application Ser. No. 11/828,275, filed Jul. 25, 2007; which is a continuation of U.S. application Ser. No. 10/830,935, filed Apr. 23, 2004; which is a continuation-in-part of U.S. application Ser. No. 10/677,404, filed Oct. 1, 2003, abandoned; which claims the benefit of provisional application 60/415,474, filed Oct. 1, 2002. The above-identified applications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for separating one or more components of interest from a sample containing particulate matter and soluble components. More particularly, the invention relates to the use of silane-treated silica filter media such as rice hull ash for separating protein and capturing particulates simultaneously. Examples of particulates include microorganisms.

BACKGROUND OF THE INVENTION

The production of materials in biotechnology involves the isolation, separation, and purification of a specific material that is surrounded by many other biological components. It does not matter whether the material comes from fermentation, a transgenic plant or the milk of a transgenic goat; the material of interest must be collected in a reasonably pure form. When the starting mixture is very complex, isolation of the material of interest can be especially difficult and often requires costly operations. Technologies that reduce the number of separation operations and simplify recovery procedures are in high demand in biotechnology and several other industries including water treatment, food and beverage, and chemicals.

Separation of product from microorganisms is important because microbial contamination is a common problem across many industries, including brewing, winery, juice and beverages, dairy, industrial enzyme and pharmaceutical. Heat sterilization and size-based filtration are by far the most commonly used processes to address this. Each of these methods has its advantages and disadvantages. The main drawback of heat sterilization is its application is limited to products that are not affected by high temperature. Sized-based filtration has the disadvantages of being expensive and time consuming. In addition, it cannot be used for processes in which the desired components are of the same size as bacteria, such as in the dairy food industry.

Examples of technologies that have been developed to simplify separations include Expanded Bed Adsorption and Chromatography. Expanded Bed Adsorption allows the capture of a soluble component from a fermentation mixture containing both soluble and particulate components. This method does not require a pre-filtration step prior to applying the sample to the bed. The fermentation mixture flows upward through a bed of adsorbent beads; the upward flow lifts and suspends the beads as the bed expands upward. The soluble components are captured by the beads while the particulate matter flows around the beads and exits the top of the bed. Then the soluble components are recovered from the beads by an elution step. This technology is not widely used yet as there are several technical hurdles including scale-up difficulty, maintaining a stable bed, carry-over of beads out of the top of the bed, fouling of the beads by the fermentation mixture, cleaning and re-use of the beads, usable life of the beads, and variable pressure drop during the course of the adsorption step.

Solid-Liquid Chromatography is any separation process that depends on solute(s) partitioning between a flowing fluid and a solid adsorbent. Many different solid adsorbents (generally referred to as “stationary-phase packing”) are used in chromatography. Different stationary-phase packings give rise to different chromatographic techniques, which are generally classified according to their mechanism of interactions. The interactions could be through one or more of the following mechanisms: charge (ion-exchange chromatography); van der Waals forces (hydrophobic interaction chromatography); size and shape (size exclusion); affinity (for example, protein-A, biotin-avidin, biotin-streptavidin, lectin, antibodies, pectin, dye ligand, immobilized metal affinity) (Reference: “Biochemical Engineering” by Harvey W. Blanch and Douglas S. Clark, Marcel Dekker Inc, 1996; p 502-506). Custom Affinity Chromatography is designed to capture a specific protein and requires a specific affinity medium with a specific ligand for each protein to be captured. Considerable time, effort, and cost are involved in developing this specific medium. In general, chromatography requires a pre-filtration step to remove solid materials.

Filtration is the removal of particulates by passing a feed stream through a porous media. Particulates are captured on the media through a variety of mechanisms including direct impaction, sieving, and others. Filtration methods employing various types of media have been used to remove particulates in such applications as wastewater treatment, winemaking, beverage making, and industrial enzyme production.

Filter media, also known as filter aids, can be loose particulate or structured material. They are solid materials in a particulate form, insoluble in the liquid to be filtered; they are added to the liquid or are coated upon a filter or filter support. The purpose of using filter media is to speed up filtration, reduce fouling of the filter surface, reduce cracking of the filter layer, or otherwise to improve filtration characteristics. Materials, which are frequently used as filter media, include cellulose fibers, diatomaceous earth, charcoal, expanded perlite, asbestos fibers and the like.

Filter media are often described according to their physical form. Some filter media are essentially discrete membranes, which function by retaining contaminants upon the surface of the membrane (surface filters). These filter media primarily operate via mechanical straining, and it is necessary that the pore size of the filter medium be smaller than the particle size of the contaminants that are to be removed from the fluid. Such a filter medium normally exhibits low flow rates and a tendency to clog rapidly.

Other filter media take the form of a porous cake or bed of fine fibrous or particulate material deposited on a porous support or substrate. The solution being filtered must wend its way through a path of pores formed in the interstices of the fine material, leaving particulate contaminants to be retained by the filter material. Because of the deepness of the filter material, the filters are called depth filters (as opposed to surface filters). Depth filters typically retain contaminants by both the sieving mechanism and the electrokinetic particle capture mechanism. In the electrokinetic particle capture mode, it is unnecessary that the filter medium have such a small pore size. The ability to achieve the required removal of suspended particulate contaminants with a filter medium of significantly larger pore size is attractive inasmuch as it allows higher flow rates. Furthermore, the filters have a higher capacity to retain particulates, thus having a reduced tendency to clog.

Biotechnology and other industries need efficient, cost-effective methods to isolate components of interest. It is also desirable to use low-cost raw materials for the process of separating biomolecules.

Rice hull ash is a byproduct of rice farming and rice is a primary food staple for half of the world's population. Currently, the inedible rice hulls are used as a source of fuel, fertilizer, and in insulation applications. When rice hulls are burned, a structured particle material having free acidic hydroxyl moieties (OH or Particle-OH) on the surface much like particle-OH of precipitated silica or fumed silica can be produced as a byproduct that has been demonstrated to be useful as a filter aid. U.S. Pat. No. 4,645,605 discloses the use of rice hull ash as filtration media.

U.S. Pat. No. 4,645,567 discloses that the filtration of fine particle size contaminants from fluids has been accomplished by the use of various porous filter media through which the contaminant fluid is passed. To function as a filter, filter media must allow the fluid (commonly water) through, while holding back the particulate. This holding back of the particulate is accomplished by distinctly different filtration mechanisms, namely (a) mechanical straining and (b) particle capturing. In mechanical straining, a particle is removed by physical entrapment when it attempts to pass through a pore smaller than itself. In particle capturing, the particle collides with a surface face within the porous filter media and is retained on the surface by short-range attractive forces.

WO 02/083270 discloses a filter system for passive filtration. The system comprising: a housing with an intake and an outlet; a pleated carbon filter disposed between the intake and the outlet for filtering out vapors entering the intake; and a hydrophobic solution including a silane composition dispersed about the pleated carbon filter to inhibit adsorption of water thereby increasing the adsorption capacity of the pleated carbon filter especially in high relative humidity environments and wherein the hydrophobic solution is selected so that it does not decrease the adsorption capacity of the carbon filter.

U.S. Pat. No. 6,524,489 discloses advanced composite media comprising heterogeneous media particles, each of said media particles comprising: (i) a functional component selected from the group consisting of diatomite, expanded perlite, pumice, obsidian, pitchstone, and volcanic ash; and (ii) a matrix component selected from the group consisting of glasses, natural and synthetic crystalline minerals, thermoplastics, thermoset plastics with thermoplastic behavior, rice hull ash, and sponge spicules; wherein said matrix component has a softening point temperature less than the softening point temperature of said functional component; and wherein said functional component is intimately and directly bound to said matrix component. The surface of the advanced composite media can be treated with dimethyldichlorosilane, hexamethyldisilazane, or aminopropyltriethoxysilane.

Snyder, et al. disclose chromatography bonded-phase packing prepared by the reaction of organosilanols, organodimethylamine, or organoalkoxy silanes with high surface area silica supports without polymerization. (L. R. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography, 2nd edition, Wiley-Interscience, N.Y. 1979. 272-280)

Roy, et al (J. Chrom. Sci. 22: 84-86 (1984)) disclose the preparation of ion-exchange (DEAE, carboxy, and sulfonic) silica using the epoxy functionality of glycidoxypropylsilyl silica; the ion-exchange silica was used in column chromatography to separated bovine serum albumin and bovine γ-globulin.

In general, treated chromatographic silica of the type described by Snyder and Roy are too expensive to be used in larger scale routine filtration and isolation processes.

There is a need for an improved and less costly separation system that is suitable for large-scale isolation of components of interest from a sample. Such a system uses low-cost raw materials and is suitable for a large-scale production and requires no pretreatment of a sample.

SUMMARY OF THE INVENTION

The present invention provides methods for separating one or more components, especially biomolecules of interest, from a sample containing particulates and soluble materials. The feature of the invention is filtering a sample through filter media whose surface has been treated by one or more silanes. Preferred filter media are silica filter media. The methods provide simultaneously capturing the particulate by filtration and binding soluble materials onto the silica filter media.

One method of the invention comprises the steps of: (a) filtering a sample through the treated silica filter media, (b) simultaneously capturing particulates and binding a soluble component of interest to the silica filter media, and (c) eluting the bound soluble component of interest from the silica filter media.

Another method of the invention comprises the steps of: (a) filtering a sample through treated silica filter media, (b) simultaneously capturing particulates and binding unwanted soluble materials to the silica filter media, (c) collecting the flow-through stream, and (d) recovering the soluble component of interest from the flow-through stream.

Another method of the invention comprises the steps of: (a) filtering a sample through treated silica filter media; (b) simultaneously removing particulate and binding a first soluble component of interest to the silica filter media, (c) collecting the flow-through stream, (d) recovering a second soluble component of interest from the flow-through stream, (e) eluting the bound first soluble component of interest from the silica filter media, and (f) recovering the first soluble component of interest.

In one embodiment of the invention, the particulates are microorganisms. In addition to being captured by the silane-treated filter media, microorganisms are also found killed by contacting with the silane-treated filter media.

The present invention is also directed to the silane-treated filter media. Preferred treated silica filter media are silane-treated rice hull ash with a functional quaternary ammonium group(s) or a functional sulphonate group(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows protein binding, and FIG. 1B shows protein release, to untreated diatomaceous earth (FW12), untreated rice hull ash, HQ50 (commercial quaternary amine anion exchange resin) and surface treated rice hull ashes (silica filter media samples 4 and 6).

FIG. 2 shows protein binding and protein release using surface-treated rice hull ashes. FIG. 2A shows the results of silica filter media samples 7 and 8. FIG. 2B shows the results of samples 9 and 10. FIG. 2C shows the results of samples 11 and 12.

FIG. 3 shows protein binding and protein release using surface-treated rice hull ashes. FIG. 3A shows the results of sample 14. FIG. 3B shows the results of silica filter media samples 13 and 15. FIG. 3C shows the results of samples 16 and 17. FIG. 3D shows the results of samples 18 and 19. FIG. 3E shows the results of sample 20.

FIG. 4 shows protein binding and protein release using surface-treated rice hull ashes. FIG. 4A shows the results of silica filter media sample 41 and untreated RHA. FIG. 4B shows the results of porous HS50.

FIG. 5 shows protein binding and protein release. FIG. 5A shows the results of silica filter media sample 42. FIG. 5B shows the results of sample 40 and untreated RHA. FIG. 5C shows the results of Celite 512. FIG. 5D shows the results of sample 29 and untreated RHA.

FIG. 6 shows dynamic protein binding and protein release using surface-treated RHA (sample 9).

FIG. 7A shows untreated rice hull ash, and FIG. 7B shows silica filter media sample 19, for simultaneous particulate filtration and soluble capture/release.

FIG. 8 shows silica filter media sample 19 and untreated RHA for simultaneous particulate filtration and soluble capture release.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for separating one or more components of interest from a sample. One embodiment of the invention comprises the steps of: (a) filtering a sample containing particulate and soluble components through silica filter media whose surface has been treated with one or more silanes, (b) simultaneously capturing particulates and binding a soluble biomolecule of interest to the silica filter media, and (c) eluting the bound soluble component of interest from the silica filter media. In this embodiment, the molecule of interest is first bound to the silica filter media and recovered later by elution. Optionally, an insoluble component of interest can be recovered from the particulates.

Another embodiment of the invention comprises the steps of: (a) filtering a sample containing particulate and soluble materials through silica filter media whose surface has been treated with one or more silanes, (b) simultaneously capturing particulates and binding unwanted soluble materials to the silica filter media, (c) collecting the flow-through stream, and (d) recovering the soluble component of interest from the flow-through stream. The soluble component of interest can be further purified from the flow-through stream. In this embodiment, the soluble component of interest does not bind to the silica filter media and is recovered in the flow-through. Optionally, an insoluble component of interest can be recovered from the particulates.

Yet another embodiment of the invention comprises the steps of: (a) filtering a sample containing particulate and soluble materials through silica filter media whose surface has been treated with one or more silanes; (b) simultaneously capturing particulates and binding a first soluble component of interest to the silica filter media, (c) collecting the flow-through stream, (d) recovering a second soluble component of interest from the flow-through stream, (e) eluting the bound first soluble component of interest from the silica filter media, and (f) recovering the first soluble component of interest. In this embodiment, the first component of interest binds to the silica filter media and the second component of interest does not bind to the silica filter media. Optionally, an insoluble component of interest can be recovered from the particulates.

The present invention optionally comprises an additional step. Prior to the filtering step (step (a)), a sample containing particulate and soluble materials first reacts with the treated silica filter media for a period of time to allow sufficient binding of the component to surface of the treated silica filter media. The reaction is carried out by mixing the sample with the treated silica filter media by any means of mechanical mixing such as agitation, stirring, vortexing, etc. After the component binds to the treated silica filter media, the mixture is applied to a filtration device and the sample is subsequently filtered through the filter media.

The term “sample” refers to any mixture containing multiple components in the form of a liquid, solution, suspension or emulsion. The sample usually includes soluble components and particulates. Of special interest are “biological samples” which refers to biological tissue and/or fluid that contains biomolecules such as polypeptides, lipids, carbohydrates, lipoproteins, polysaccharides, sugars, fatty acids, polynucleotides, or viruses. A biological sample may contain sections of tissues such as frozen sections taken for histological purposes. A sample suitable for this invention includes cell lysate, culture broth, food products and particularly dairy products, blood, beverages (for example, juice, beer, wine), and a solution or a suspension containing biomolecules such as proteins. “Proteins” are natural, synthetic, and engineered peptides or polypeptides, which include enzymes such as oxidoreductases, transferases, isomerases, ligases, and hydrolases, antibodies, hormones, cytokines, growth factors, and other biological modulators.

Filtration is the removal of particulates by passing a feed stream through a porous media. Particulates are captured on the media through a variety of mechanisms including physical entrapment, and binding to the media. The present invention utilizes silica media filter of various types to remove particulates in different applications, including wastewater treatment, winemaking, juice and beverage making, diary, and industrial production of proteins such as enzymes.

The term “particulates” refers to macroscopic insolubles or microscopic particulates. Macroscopic particulates are those that are visible to the human eye, including, but not limited to precipitates, inclusion bodies, and crystals. Inclusion bodies consist of insoluble and incorrectly folded protein in the cellular compartment. Crystals are formed from supersaturated solutions by aggregation of molecules, occurring in an ordered, repetitive fashion. Precipitates are amorphous form from random aggregation. Macroscopic particulates can be of organic or inorganic origin; they can be derived from the interaction between protein and protein, salt and protein, salt and salt, protein and polymer, etc. Microscopic particulates are those that can be seen under a microscope. Examples of particulates include microorganisms. Microorganisms suitable to be captured and removed from a biological sample by the present invention are gram-positive bacteria, gram-negative bacteria, fungi, yeast, mold, virus, etc.

The feature of this invention is using treated silica filter media in a filtration process to simultaneously bind soluble components onto the silica filter media and capture particulates from a solution by filtration. The present invention eliminates a pre-filtration step often required in a chromatography process to remove particulate. Soluble components bind to the silane-treated silica filter media through different mechanisms such as hydrophilic, hydrophobic, affinity and/or electrostatic interactions. Silica filter media useful for this invention have surfaces suitable for treatment with silanes and structural characteristics suitable for industrial filtration applications. Examples of silica filter media include, but are not limited to, rice hull ash, oat hull ash, diatomaceous earth, perlite, talc, and clay.

Rice hull ash is a byproduct of rice farming. Each grain of rice is protected with an outer hull, which accounts for 17-24% of the rough weight of the harvested product. Rice hulls consist of 71-87% (w/w) organic materials, such as cellulose and 13-29% (w/w) inorganic materials. A significant portion of the inorganic fraction, 87-97% (w/w) is silica (SiO₂). Currently, the inedible rice hulls are used as a source of fuel, fertilizer, and in insulation applications. When the rice hulls are burned, a structured silica material (often greater than 90%) can be produced as a byproduct. Rice hull ash (RHA) has larger surface area and more porous-channeled structure compared with other loose silica filter media. These characteristics make the RHA a preferred treated filter substrate for this invention.

Diatomaceous earth (Diatomite) is a sedimentary silica deposit, composed of the fossilized skeletons of diatoms, one celled algae-like plants which accumulate in marine or fresh water environments. The honeycomb silica structures give diatomite useful characteristics such as absorptive capacity and surface area, chemical stability, and low bulk density. Diatomite contains 90% SiO₂ plus Al, Fe, Ca and Mg oxides.

Perlite is a generic term for a naturally occurring siliceous volcanic rock that can be expanded with heat treatment. Expanded perlite can be manufactured to weigh as little as 2 pounds per cubic foot (32 kg/m³). Since perlite is a form of natural glass, it is classified as chemically inert and has a pH of approximately 7. Perlite consists of silica, aluminum, potassium oxide, sodium oxide, iron, calcium oxide, and magnesium oxide. After milling, perlite has a porous structure that is suitable for filtration of coarse microparticulates from liquids it is suitable for depth filtration.

Talc (talcum) is a natural hydrous magnesium silicate, 3 MgO.4SiO₂.H₂O. Clay is hydrated aluminum silicate, Al₂O₃.SiO₂.xH₂O. Mixtures of the above silica filter media substrates can also be used to achieve the best filtration and cost performance. The rice hull ash or diatomaceous earth has optionally undergone various purification and/or leaching steps before the surface silane treatment.

Silica filter media are treated by binding a predetermined amount of functional silane (or silanes) to the surface. The treated silica filter media capture components, for example, by electrostatic, hydrophilic, hydrophobic, affinity interactions, and/or by physical entrapment. By electrostatic interaction, the charged silica filter media bind to materials in a sample that have the opposite charge. By hydrophilic interaction, the portion of the silica filter media that has a strong affinity for water attracts the polar group of the materials by van der Waals interaction. By hydrophobic interaction, the portion of the silica filter media that contains long hydrocarbon chains attracts the non-polar groups of the materials. The treated silica filter media selectively capture materials (desired or undesired) during the separation process, which results in better separation characteristics comparing with non-treated silica filter media. The treated silica filter media preferably have a similar or improved flow rate compared with the non-treated silica filter media.

The form of silica filter media substrate materials can be any form suitable for the application, such as spheres, fibers, filaments, sheets, slabs, discs, blocks, films, and others. They can be manufactured into cartridges, disks, plates, membranes, woven materials, screens etc. The specific surface area of the untreated silica filter media is preferred to be larger than 1 m²/g; more preferred to be larger than 10 m²/g. Silica filter media with a larger surface area are preferable because they allow more treatment on the surface. In addition, media with large pores improve the filtration rate. However, larger pore materials have relatively lower surface area. The balance of large surface area and large pore size results in effective surface filtration treatment and filtration rate. The surface characteristics of these substrates can be evaluated by techniques such as NMR (Nuclear Magnetic Resonance and other techniques), SEM (Scanning Electron Microscopy), BET (Brunauer-Emmett-Teller) surface area measurement technique, and Carbon-hydrogen-nitrogen content can be determined by combustion techniques, which are well known to the art.

Silanes suitable for surface treatment of silica filter media can be any type of organosilanes, ionic or non-ionic. The general formula of the suitable silane is (R¹)_(x)Si(R²)_(3-x)R³,

wherein R¹ is typically a hydrolysable moiety (such as alkoxy, halogen, hydroxy, aryloxy, amino, amide, methacrylate, mercapto, carbonyl, urethane, pyrrole, carboxy, cyano, aminoacyl, or acylamino, alkyl ester, or aryl ester), which reacts with the active group on the silica filter media; a preferred hydrolysable moiety is alkoxy group, for example, a methoxy or an ethoxy group;

1≦x≦3, more than one siloxane bond can be formed between the filter particle and silane;

R² can be any carbon-bearing moiety that does not react with the filter surface during treatment process, such as substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl;

R³ can be any organic containing moiety that remains chemically attached to the silicon atom once the surface reaction is completed, and preferably it can interact with the component of interest during filtration; for example R₃ is hydrogen, alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, arylakaryl, alkoxy, halogen, hydroxy, aryloxy, amino, amide, methacrylate, mercapto, carbonyl, urethane, pyrrole, alkyl ester, aryl ester, carboxy, sulphonate, cyano, aminoacyl, acylamino, epoxy, phosphonate, isothiouronium, thiouronium, alkylamino, quaternary ammonium, trialkylammonium, alkyl epoxy, alkyl urea, alkyl imidazole, or alkylisothiouronium;

wherein the hydrogen of said alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic is optionally substituted by halogen, hydroxy, amino, carboxy, or cyano.

One or more silanes can be covalently bound to the surface of the hydroxyl bearing porous silica filter media. The surface area of the silica filter media limits the amount of the silanes bound.

Silanes useful for treating silica in this invention preferably have one or more moieties selected from the group consisting of alkoxy, quaternary ammonium, aryl, epoxy, amino, urea, methacrylate, imidazole, carboxy, carbonyl, isocyano, isothiorium, ether, phosphonate, sulfonate, urethane, ureido, sulfhydryl, carboxylate, amide, carbonyl, pyrrole, and ionic. Examples for silanes having an alkoxy moiety are mono-, di-, or trialkoxysilanes.

Examples of silanes having a quaternary ammonium moiety are 3-(trimethoxysilyl)propyloctadecyldimethylammoniumchloride, N-trimethoxysilylpropyl-N,N,N-trimethylammoniumchloride, or 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride. Examples of silanes having an aryl moiety are 3-(trimethoxysilyl)-2-(p,m-chlandomethyl)-phenylethane, 2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone, ((chloromethyl)phenylethyl)trimethoxysilane and phenyldimethylethoxysilane. Examples of silanes having an epoxy moiety are 3-glycidoxypropyltrimethoxysilane and 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. Examples of silanes having an amino moiety are 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, trimethoxysilylpropyldiethylenetriamine, 2-(trimethoxysilylethyl)pyridine, N-(3-trimethoxysilylpropyl)pyrrole, trimethoxysilylpropyl polyethyleneimine, bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane, and bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

Examples of silanes having an urea moiety are N-(triethoxysilylpropyl)urea and N-1-phenylethyl-N′-triethoxysilylpropylurea. An example of silanes having a methacrylate moiety is 3-(trimethoxysilyl)propyl methacrylate. An example of silanes having a sulfhydryl moiety is 3-mercaptopropyltriethoxysilane. Examples of silanes having an imidazole moiety are N-[3-(triethoxysilyl)propyl]imidazole and N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole. Examples of ionic silanes are 3-(trimethoxysilyl)propyl-ethylenediamine triacetic acid trisodium salt; and 3-(trihydroxysilyl)propylmethylposphonate sodium salt. An examples of silanes having a carbonyl moiety is 3-(triethoxysilyl)propylsuccinic anhydride. Examples of silanes having an isocyano moiety are tris(3-trimethoxysilylpropyl)isocyanurate and 3-isocyanatopropyltriethoxysilane. Examples of silanes having an ether moiety are bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane.

An example of a silane having a sulfonate moiety is 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane. An example of a silane having a isothiourium moiety is trimethoxysilylpropylisothiouronium chloride. Examples of silanes having an amide moiety are triethoxysilylpropylethyl-carbamate, N-(3-triethoxysilylpropyl)-gluconamide, and N-(triethoxysilylpropyl)-4-hydroxybutyramide. Examples of silanes having a urethane moiety are N-(triethoxysilylpropyl)-O-polyethylene oxide urethane and O-(propargyloxy)-N-(triethoxysilylpropyl)urethane.

Silica filter media can also be treated with more than one silanes such as 3-aminopropyltrimethoxysilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 3-trihydrosilylpropylmethylphosphonate, sodium salt and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and (3-glycidoxypropyl)trimethoxysilane; 3-trihydrosilylpropylmethylphosphonate, sodium salt and bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane; 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 2-(trimethoxysilylethyl)pyridine and N-(3-triethoxysilylpropyl)-gluconamide; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and N-(3-triethoxysilylpropyl)-gluconamide; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and 2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone; 3-mercaptopropyltriethoxysilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 3-(triethoxysilyl)propylsuccinic anhydride and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium salt and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; and 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane and bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

The silane-treated silica filter media have a general formula selected from the group consisting of particle-O—Si(R¹)_(x)(R²)_(3-x)R³,

wherein R¹, R², R³, and x are the same as described above so long as there are no more than four groups directly attached to the silicon (Si);

R⁵, R⁶, R⁸ are independently hydrogen, substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, ether, ester or arylalkaryl;

R⁴, R⁷, R⁹ are substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl radicals capable of forming two covalent attachments.

The silica filter media with surface silanol are treated with silane in a general reaction scheme as following:

Particle-OH+(R¹)_(x)Si(R²)_(3-x)R³→Particle-O—Si(R¹)_(3-n)(R²)_(3-x)R³ +nR¹H

where Particle-OH is a filter particle with reactive sites on surface. For example, R¹ is a methoxy (CH₃O—) or ethoxy (CH₃CH₂O—) labile leaving group of the silane, which chemically interacts, with the reactive hydroxyl group on the particle surface or with other reactive hydrolyzed silane molecules which are not attached to the surface. 1≦x≦3; n is the number of R¹ groups reacted, and n≦x.

Prolonged reaction of excess amounts of reactive silane under anhydrous conditions results in reaction of only 25% to 50% of the total active sites on the porous material since further reaction is inhibited by steric hindrance between the immobilized residues and is also hindered by access to deeply imbedded Particle-OH groups. For the purposes of this invention, such sterically available sites will be designated as the “saturation coverage” and “saturation coverage” depends upon the steric requirements of a particular residue. Note that this designation of “saturation coverage” is applicable to reactive silanes with one or more labile leaving groups. Under anhydrous conditions, such silanes form monolayers and cannot form multiple layers of undefined saturation. However, under aqueous conditions, multiple layers can be built on the surface with multifunctional silanes.

The surface silane treatment of silica filter media can be carried out by an essentially “wet” or essentially “dry” process. The essentially wet process consists of reacting the silane onto the silica filter media in a solvent (organic solvent or water) and optionally using heat. Heat or solvent is not required for the reaction; however, heat or solvent improves the reaction rate and the uniform surface coverage. The essentially dry process consists of reacting the silane onto the silica filter media in a vapor phase or highly stirred liquid phase by directly mixing the silane with silica filter media and subsequently heating.

A preferred method for treating silica filter media with silanes is adding the reacting silanes gradually to a rapidly stirred solvent, which is in direct contact with the porous silica filter media. Another preferred method is to carry out the treatment in the vapor phase by causing the vapor of the reactive silanes to contact and react with the silica filter media. For example, the porous material is placed in a vacuum reactor and dried under vacuum. The rapidly reacting silane is then allowed to enter the vacuum chamber as a vapor and contact the porous material; after a certain contact time, the byproducts of the reaction are removed under reduced pressure. Then the vacuum is released, and the porous material is removed from the chamber.

The actual treatment process can be carried out in a period from 1 minute to 24 hours. Generally, for purposes of this invention, it is preferred to carry out the treatment over a period from about 30 minutes to 6 hours to ensure that the surface of the filter aid material is uniformly treated. The treatments are carried out at temperatures ranging from 0 to 400° C. Preferred treatment temperatures are from room temperature (22 to 28° C.) to 200°.

The amount of reacting silanes used in this invention depends on the number of surface hydroxyls to be reacted, and the molecular weight of the silane. Typically, a stoichiometric amount equivalent to the available surface hydroxyls plus some excess amount of the reacting silane is used to treat the surface hydroxyls because of the potential side reactions. If a thicker exterior surface treatment is desired, then more reacting silane should be used. Typically, 0 to 10 (preferred), 0 to 20, or 1 to 50 times excess is used. However, it is not uncommon to use from 1 to 500 times excess; which results in more treatment on the particle.

Silanes with hydrolysable groups condense with Particle-OH groups of the surface of the particles, and provide covalent coupling of organic groups to these substrates. For example, the alkoxy groups of the silanes chemically react with the Particle-OH groups of the particle surface. The surface-silane interaction is fast and efficient. For example, when silanes having a quaternary ammonium moiety are used, the protonated positively charged silanes electrostatically attract to the deprotonated groups of the particle efficiently to facilitate fast and efficient reaction.

Silane-reacted silica filter media preferably have a functional moiety, which can react with a component of interest. The functional moiety is selected from the group consisting of quaternary ammonium, epoxy, amino, urea, methacrylate, imidazole, sulphonate and other organic moieties known to react with biological molecules. In addition, the functionally moiety can be further reacted, using well-known methods, to create further new functionalities for other interactions. General schemes for preparation of a silane-reacted particle filter media with a functional quaternary ammonium or sulphonate group are illustrated as follows.

Silane-reacted particle filter media with a functional quaternary ammonium group can be prepared in one step. Optionally, a two step or three step process may be employed. For example, in the first step of the two step process, the particle surface is reacted with an amino-functional silane, (R¹)_(X)Si(R²)_(3-X)R⁴N(R⁵)₂, applying the previously described procedure. In the next step, the secondary amine readily reacts with the epoxide group of the glycidyltrimethylammoniumchloride, which is a convenient way to introduce quaternary ammonium functionality. (See Scheme 1)

Silane-reacted silica filter media with a functional sulphonate group can be prepared in two steps. In the first step, the particle surface is reacted with an epoxy-functional silane applying the previously described procedure. In the next step, the epoxy functionality readily reacts with sodium bisulfate to produce sulphonate-functional silica filter media. (See Scheme 2). Sodium metabisulfite (Na₂S₂O₅) decomposes in water to form sodium bisulfate (NaHSO₃).

The silane-treated particles are used in separation applications to capture soluble materials through electrostatic, and/or hydrophobic, and/or hydrophilic interaction mechanisms while removing particulates. The advantage of the treated silica filter media is that the separation process is simplified by combining the filtration and solid phase extraction in a single step. The desired quality of the treated silica filter media is to have similar or improved flow rate (filtration properties) to the untreated silica filter media along with the capability to capture soluble materials through sorption in a single operation.

In one embodiment of the invention, specific charged groups are attached covalently to the surface of the silica particles to capture materials electrostatically. The oppositely charged materials are bound to the porous treated surface. In addition to the electrostatic attraction, hydrophobic or hydrophilic ligands are used to improve the binding and/or release characteristics of the silica filter media by hydrophobic or hydrophilic interaction.

Treated silica filter media are characterized by measuring surface area, pore volume and pore size using methods known to the art such as a Micrometrics® analyzer. For example, surface area can be characterized by BET technique. Pore volume and pore diameter can be calculated by Barrett-Joyner-Halenda analysis. Specific functional groups and molecular structure can be determined by NMR spectroscopy. Carbon-hydrogen-nitrogen content can be determined by combustion techniques; from this analysis information, the treatment level on the particle surface can be calculated.

A sample, such as fermentation broth, can be applied to silane-treated silica filter media without pre-filtration. In one embodiment, the sample is mixed with the treated silica filter media by any means of mechanical mixing (such as agitation, stirring, vortexing, etc.) for a period of time to allow sufficient binding of the component to the surface of treated silica filter media. Those skilled in the art will recognize that the time suitable for binding is dependent upon the character of the pores of the media, the size of the target molecule or component, the viscosity and other known mass transfer limited principles. Generally, the time for binding to occur varies from about a few minutes to a few hours, but may continue up to 1-3 days. The mixture is then filtered using a filtration unit. In another embodiment, a sample can be filtered directly through a filtration unit containing silane-treated silica filter media without pre-mixing the sample with the filter media. The treated silica filter media capture particulates and bind certain soluble components while allowing the unbound soluble components to flow through. The bound component is extracted by flowing an eluting solution through the filtration unit, and is recovered in an eluate stream. Useful eluting solutions include salt solutions, high pH (basic) solutions, low pH (acidic) solutions, chaotropic salts and other reagents. Alternately, common organic solvents or mixtures thereof may be used as long as they do not have deleterious affects on the recovered component of interest. Suitable high salts include NaCl, KCl, LiCl, etc. Suitable chaotropic salts include sodium perchlorate, guanidine hydrochloride, guanidine isothiocyanate, potassium iodide, etc. Other reagents include urea and detergents. Combinations of the above components are also suitable in some applications. Alternately, an eluting solution is used to resuspend the surface silica filter media (containing particulates and bound molecules) by any means of mechanical mixing for a period of time to allow sufficient release of the bound component before filtering.

One application of the invention is to use the silane-treated silica filter media to separate microorganisms from a desired product. Microbial contamination is a common problem across many industries, including brewing, winery, juice and beverages, dairy, industrial enzyme and pharmaceutical. Applicants have found that the silane-treated silica filter media of this invention have anti-microbial activity. By contacting bacteria with the silane-treated silica filter media, the total viable bacterial counts are significantly reduced. The microorganisms are also captured by the silane-treated silica filter media. Thus, the filtration step further removes the microbial contamination from the product.

The present invention is also directed to a silane-treated silica filter media having a general formula selected from the group consisting of particle-O—Si(R¹)_(x)(R²)_(3-x)R³,

wherein R¹ is alkoxy, halogen, hydroxyl, aryloxy, amino, carboxy, cyano, aminoacyl, or acylamino, alkyl ester, or aryl ester;

R² are independently substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl;

R³ is hydrogen, alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, arylakaryl, alkoxy, halogen, hydroxyl, aryloxy, amino, alkyl ester, aryl ester, carboxy, sulphonate, cyano, aminoacyl, acylamino, epoxy, phosphonate, isothiouronium, thiouronium, alkylamino, quaternary ammonium, trialkylammonium, alkyl epoxy, alkyl urea, alkyl imidazole, or alkylisothiouronium; wherein the hydrogen of said alkyl, alkenyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclic is optionally substituted by halogen, hydroxyl, amino, carboxy, or cyano;

R⁵, R⁶ and R⁸ are independently hydrogen, substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl;

R⁴, R⁷, R⁹ are substituted or unsubstituted alkyl, alkenyl, alkaryl, alkcycloalkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, cycloalkaryl, cycloakenylaryl, alkcycloalkaryl, alkcycloalkenyaryl, or arylalkaryl radicals capable of forming two covalent attachments;

wherein said silica filter media is rice hull ash or oat hull ash.

The silane-reacted silica filter media of the present invention preferably have a functional moiety, which can react with a component of interest. The functional moiety is selected from the group consisting of quaternary ammonium, epoxy, amino, urea, methacrylate, imidazole, sulphonate and other organic moieties known to react with biological molecules.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. Examples 1 through 5 illustrate the surface treatment of silica filter media. Examples 6 through 14 illustrate the use of the silane treated filter media for separating one or more components of interest from a sample containing particulate matter and soluble components. Examples 15-22 illustrate the antimicrobial activity of the silane-treated silica filter media and the filtration results.

EXAMPLES Example 1 Preparation of Treated Rice Hull Ash Media (tRHA) Using Trialkoxysilanes in a Batch Process

The treatment equipment is composed of a 3-neck, round bottom reaction flask, a Teflon shaft mechanic stirrer, thermometer, condenser, and heating mantle around the flask. The reaction flask was loaded with ungrounded RHA silica filter media (surface area: ˜30 m²/g), and solvent mixture. Table 1 shows the reaction conditions for each example. The mixture was stirred for a few minutes at ambient temperature, then the surface modification process involved addition of the proper amount of the silane directly to the mixture in a slow addition rate, while good mixing was maintained. 250% of the proper amount of the silane as calculated to achieve multilayer coverage (high-level treatment) or 85% of the amount of silane as calculated to achieve monolayer coverage (low level treatment) was added and the silane quantity was also corrected for their purity. The loading concentrations are also listed in Table 1. Subsequently, the mixture was heated to reflux under N₂ blanket, which is used primarily for safety and has no other affect on the outcome of the reaction, although heating is not required. After 2 hours stirring and refluxing, the treated slurry mixture was allowed to cool. Then it was transferred to a porcelain Büchner funnel outfitted with Whatman filter paper, and attached to a vacuum filter flash. The treated filter slurry was filtered and washed twice with toluene and twice with IPA. Afterward, the sample was dried in the hood for about 24 hours. The treated filter media was transferred to a Pyrex container and covered with a paraffin film having a number of holes made with a syringe needle, and then the sample was dried in a vacuum oven at 60° C. for 2-4 hours. The dried samples were analyzed for surface area, pore structure, and carbon-hydrogen-nitrogen content.

TABLE 1 Summary of treatment compositions and conditions Treatments are done on low carbon, ungrounded RHA from Producers. Silica Silane Added Sample # Amount g Silane Type Treatment Condition Purity % Silane g 1 50 3-(trimethoxysilyl)propyloctadecyl- H₂O, reflux 42% 15.06 dimethylammonium chloride 2 50 3-(trimethoxysilyl)propyloctadecyl- H₂O, room temp. 42% 15.06 dimethylammonium chloride 3 50 3-(trimethoxysilyl)propyloctadecyl- Toluene, reflux, 42% 15.06 dimethylammonium chloride stochiometric H₂O 4 50 3-(trimethoxysilyl)propyloctadecyl- Toluene, IPA, reflux 42% 15.06 dimethylammonium chloride 5 50 3-(trimethoxysilyl)propyloctadecyl- Toluene, IPA, reflux, 42% 15.06 dimethylammonium chloride stochiometric H₂O at end 6 50 3-(trimethoxysilyl)propyloctadecyl- Toluene, IPA, reflux 42% 7.03 dimethylammonium chloride 7 50 3-(trimethoxysilyl)-2-(p,m-chloromethyl)- Toluene, IPA, reflux 90% 1.47 phenylethane 8 50 3-(trimethoxysilyl)-2-(p,m-chloromethyl)- Toluene, IPA, reflux 90% 4.33 phenylethane 9 50 3-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 32% 13.30 propyltrimethoxysilane hydrochloride 10 50 3-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 32% 4.99 propyltrimethoxysilane hydrochloride 11 50 N-trimethoxysilylpropyl-N,N,N- Toluene, IPA, reflux 50% 7.32 trimethylammonium chloride 12 50 N-trimethoxysilylpropyl-N,N,N- Toluene, IPA, reflux 50% 2.49 trimethylammonium chloride 13 50 3-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 40% 6.69 propyltrimethoxysilane hydrochloride 14 50 3-(N-styrylmethyl-2-aminoethylamino)- Toluene, IPA, reflux 40% 19.67 propyltrimethoxysilane hydrochloride 17 100 3-aminopropyltrimethoxysilane Toluene, IPA, reflux 100% 7.52 18 100 3-aminopropyltrimethoxysilane Toluene, IPA, reflux 100% 2.56 19 100 N-(2-aminoethyl)-3- Toluene, IPA, reflux 97% 9.62 aminopropyltrimethoxysilane 20 100 N-(2-aminoethyl)-3- Toluene, IPA, reflux 97% 3.27 aminopropyltrimethoxysilane 21 50 Phenyldimethylethoxysilane Toluene, IPA, reflux 100% 1.82 22 50 Phenyldimethylethoxysilane Toluene, IPA, reflux 100% 0.76 23 50 3-(trimethoxysilyl)propyl methacrylate Toluene, IPA, reflux 98% 7.66 24 50 N-(triethoxysilylpropyl)urea Toluene, IPA, reflux 49% 5.44 25 50 Trimethoxysilylpropyldiethylenetriamine Toluene, IPA, reflux 98% 2.73 26 50 Bis(2-hydroxyethyl)-3- Toluene, IPA, reflux 58% 4.96 aminopropyltriethoxysilane 27 50 N-[3-(triethoxysilyl)propyl]imidazole Toluene, IPA, reflux 96% 2.88

Example 2 Preparation of Different Types of Treated Silica Filter Media

Additional substrates, namely high carbon rice hull ash, different types of ultra pure diatomaceous earth (Celpure P1000, Celpure P65), Celite 545 (standard diatomaceous earth filter aid), Perlite, and LRA II (a non-silica based lipid adsorbent) were used. Table 2 summarizes the treatment conditions and compositions of these samples.

TABLE 2 Compositions and conditions of treatments of different substrates Loading Silica Substrate Silica Treatment Silane (X Monolayer Added Sample # Media Type Amount g Treatment Type Condition Purity % coverage) Silane g 28 AgriSilicas 150 AEAPTMS Toluene, IPA, 97% 150% 10.53 STD (A 0700) reflux 29 Celpure 100 AEAPTMS Toluene, IPA, 97% 180% 0.51 P1000 (A 0700) reflux 30 Celpure 50 AEAPTMS Toluene, IPA, 97% 1070% 1.53 P1000 (A 0700) reflux 31 Celpure 50 Z-6032 Toluene, IPA, 32% 200% 1.46 P1000 (SMAEAPTMS) reflux 32 Perlite 50 AEAPTMS Toluene, IPA, 97% 200% 0.24 (A 0700) reflux 33 Perlite 50 Z-6032 Toluene, IPA, 32% 200% 1.21 (SMAEAPTMS) reflux 34 Celite 545 50 AEAPTMS Toluene, IPA, 97% 200% 0.40 (A 0700) reflux 35 Celite 545 50 Z-6032 Toluene, IPA, 32% 200% 2.05 (SMAEAPTMS) reflux 36 Celpure P65 50 AEAPTMS Toluene, IPA, 97% 200% 0.61 (A 0700) reflux 37 Celpure P65 50 Z-6032 Toluene, IPA, 32% 200% 3.13 (SMAEAPTMS) reflux 38 LRA II 50 AEAPTMS Toluene, IPA, 97% 120% 8.96 (A 0700) reflux 39 LRA II 50 Z-6032 Toluene, IPA, 32% 120% 45.80 (SMAEAPTMS) reflux Z-6032: 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride AEAPTMS: N-(2-aminoethyl)-3-aminopropyltrimethoxysilane

Example 3 Two-Step Process to Synthesize Hydrophilic Quaternary Ammonium Functional Filter Aids (Filter Media Samples 40 and 42)

The treatment equipment was composed of a 500 milliliter, 3-neck round bottom reaction flask, a Teflon shaft mechanic stirrer, thermometer, condenser, and heating mantle around the flask. The reaction flask was loaded with 50 g of amino-functional pretreated RHA (sample 17 or 19) silica filter media, and 200 ml IPA solvent. The mixture was stirred for few minutes at ambient temperature, then the surface modification process involved addition of the proper amount of glycidyltrimethylammonium chloride (2.46 g for sample 17, or 2.02 g for sample 19) directly to the mixture in a slow addition rate, while good mixing was maintained. The reaction mixture was heated and refluxed under a N₂ blanket. After 4 hours stirring and refluxing, the treated slurry mixture was allowed to cool. Then it was transferred to a porcelain Büchner funnel outfitted with Whatman filter paper, and attached to a vacuum filter flask. The treated filter cake was filtered and washed four times with about 150 ml of DI water each time. Afterward, the sample was dried in the hood for about 24 hours. Next the treated silica filter media was transferred to a Pyrex container and covered with a paraffin film having a number of holes made with a syringe needle, and then the sample was vacuum oven dried at 60° C. for 2-4 hours. The dried samples were analyzed for surface area, pores structure, carbon-hydrogen-nitrogen content, ²⁹Si NMR.

Example 4 Two-Step Process to Synthesize Hydrophilic Sulphonate-Functional Filter Aids (Filter Media Sample 41)

The treatment equipment was composed of a 500 milliliter, 3-neck round bottom reaction flask, a Teflon shaft mechanic stirrer, thermometer, condenser, and heating mantle around the flask. The reaction flask was loaded with 50 g of epoxy-functional pretreated RHA silica filter media (sample 15), and 200 ml IPA:H₂O (5:1) solvent. The mixture was stirred for few minutes at ambient temperature, and the reaction mixture heated up to 70° C. under a N₂ blanket. The surface modification process involved addition of the mixture of 0.55 g of sodium metabisulfite, 0.07 g of sodium sulfite catalyst, and 5 g water from an additional funnel directly to the mixture in a slow addition rate over 1-2 hours, while good mixing was maintained. The temperature was then raised up to approximately 80° C., until the reaction completed. The reaction was monitored by iodometric titration of residual NaHSO₃. After approximately 22 hours stirring and refluxing, the treated slurry mixture was allowed to cool. Then it was transferred to a porcelain Büchner funnel outfitted with Whatman filter paper, and attached to a vacuum filter flask. The treated filter cake was filtered and washed four times with about 150 ml of DI water each time. Afterward, the sample was dried in the hood for about 24 hours. Next the treated filter aid was transferred to a Pyrex container and covered with a paraffin film having a number of holes made with a syringe needle, and then the sample was vacuum oven dried at 60° C. for 2-4 hours. The dried samples were analyzed for surface area, pores structure, carbon-hydrogen-nitrogen content, ²⁹Si NMR. Table 3 summarizes compositions and conditions of the two-step processes.

TABLE 3 Compositions and conditions of treatments of two step processes. Silica Silane Added Sample # Amount g 2nd Step Reactant Treatment Condition Purity % Silane g 40 50 Glycidyltrimethylammonium chloride IPA, reflux 75% 2.02 41 50 Na₂S₂O₅/Na₂SO₃ IPA, water, reflux 100% 0.55/0.07 42 50 Glycidyltrimethylammonium chloride IPA, reflux 75% 2.46

Characterization of the Treated Silica Filter Media: BET Surface Area, Pore Volume, Pore Diameter

The surface area and porosity were measured using a Micrometrics® ASAP 2010 analyzer. Before analyses, the samples were degassed under vacuum at 150° C. until a constant pressure was achieved. In the analysis step, N₂ gas was adsorbed by the sample at 77° K. and the surface area was calculated from the volume of adsorbate. BET parameters were acquired by integration of the BET equation using ASAP-2010 software. Surface area was calculated in the range of 0.05≦P/Po≦0.3 from the adsorption branch of the isotherm. Barrett-Joyner-Halenda analysis was used to calculate the pore volume and pore diameter.

NMR

Identification of specific functional groups and molecular structure was determined by ²⁹Si solid state NMR spectroscopy on a Unity Plus 400 MHz Spectrometer using a Varian VT CPMAS probe and a 7 mm motor.

Carbon-Hydrogen-Nitrogen (CHN)

CHN content was determined by combustion technique at Robertson Microlit Laboratories. From this analysis information, the treatment level on the surface was calculated.

Table 4 summarizes the characterization data of the treated silica samples.

TABLE 4 Characterization data summary of treated silica samples % C Surface Pore by Moisture Area Volume Pore Robertson Ligand density Sample # content % m²/g cm³/g Diameter Å Microlit μmol/m² μmol/g 1 2.63% 8.69 0.047 149.54 5.69% 23.73 206.16 2 4.43% 11.58 0.060 142.22 5.58% 17.47 202.17 3 2.05% 17.85 0.077 98.42 5.12% 10.39 185.51 4 1.60% 22.51 0.097 97.05 3.11% 4.61 103.67 5 1.43% 23.45 0.098 93.15 2.96% 4.57 107.25 6 1.89% 24.53 0.104 94.57 2.47% 3.36 82.33 7 1.57% 32.65 0.128 99.68 0.84% 1.95 63.64 8 2.60% 33.66 0.129 99.64 1.01% 2.27 76.52 9 2.20% 22.98 0.101 105.56 2.19% 4.96 114.06 10 1.46% 29.32 0.118 96.80 1.32% 2.35 68.75 11 1.33% 30.24 0.124 100.45 1.67% 5.75 173.96 12 1.44% 22.39 0.103 112.07 0.88% 4.09 91.67 13 1.59% 28.19 0.112 95.47 2.09% 3.86 108.85 14 1.77% 18.76 0.077 101.39 2.98% 8.27 155.21 17 2.71% 28.02 0.100 97.28 1.36% 8.09 226.67 18 0.86% 30.48 0.118 100.00 0.72% 3.94 120.00 19 1.49% 23.64 0.101 101.93 1.68% 8.46 200.00 20 1.75% 28.15 0.118 98.55 1.03% 4.36 122.62 21 1.44% 32.32 0.131 102.99 0.42% 1.35 43.75 22 2.47% 32.28 0.133 104.50 0.23% 0.74 23.96 23 0.80% 29.80 0.120 97.08 0.98% 3.04 90.74 24 1.05% 28.99 0.119 100.14 0.80% 2.87 83.33 25 2.06% 27.02 0.117 100.15 1.14% 3.91 105.56 26 0.96% 31.75 0.128 100.93 0.74% 1.77 56.06 27 1.63% 31.06 0.129 102.94 0.62% 1.66 51.67 28 2.90% 16.11 0.023 215.71 0.82% 6.06 97.62 29 0.33% 2.18 0.002 106.61 0.09% 4.92 10.71 31 0.04% 2.39 0.003 140.36 0.46% 10.02 23.96 33 5.68% 3.07 0.003 148.64 0.57% 9.66 29.69 34 0.48% 1.47 0.002 104.07 0.16% 12.94 19.05 35 0.05% 2.11 0.002 139.39 0.22% 5.42 11.46 37 0.94% 5.66 0.014 145.31 0.39% 3.59 20.31 39 5.31% 112.73 0.741 222.48 8.71% 4.02 453.65 40 2.77% 21.82 0.099 105.43 1.82% 5.35 116.67 41 2.69% 29.02 0.114 98.12 0.99% 3.55 103.13 42 1.91% 26.17 0.109 102.99 1.41% 4.08 106.82

Example 5 Compositions and Treatment Conditions of Silica Filters and their Characterization

Table 5A-5C summarized additional compositions and treatment conditions of rice hull ash and their characterization.

TABLE 5A Treatment Preparation Filter Reagent Me- gram Results dia Glycidyl- Density Sam- trimethyl- Ligand ple Treatment Silica Silica First Additive Second Additive ammonium % C μmol/ No Type Type gram Name Gram Name Gram chloride % m² 43 3-(N-styrylmethyl-2- Producers 25 3-(N-styrylmethyl-2- 19.83 5.61% 25.68 aminoethylamino)- RHA aminoethylamino)- propyltrimethoxy- propyltrimethoxy-silane silane hydrochloride hydrochloride 44 3-(Trimethoxysilyl Producers 25 3-(Trimethoxysilyl 3.88 1.06% 11.34 propyl) RHA propyl) isothiouronium isothiouronium chloride chloride 45 3-(Trimethoxysilyl- Producers 25 3- 3.88 1.67% 18.27 propyl) RHA (Trimethoxysilylpropyl) isothiouronium isothiouronium chloride chloride 46 N-Octadecyldimethyl RiceSil 500 N- 93.29 N- 4.12 2.46% 5.41 (3-Trimethoxysilyl 100 Octadecyldimethyl(3- (Triethoxysilyl- propyl) Trimethoxysilylpropyl) propyl)-o- ammonium chloride, ammonium chloride polyethylene then N- oxide urethane (Triethoxysilyl- propyl)-o- polyethylene oxide urethane 47 3-(N-styrylmethyl-2- RiceSil 500 3-(N-styrylmethyl-2- 92.66 N- 3.12 1.96% 6.37 aminoethylamino)- 100 aminoethylamino)- (Triethoxysilyl- propyl propyltrimethoxysilane propyl)-o- trimethoxysilane hydrochloride polyethylene hydrochloride, then oxide urethane N- (Triethoxysilylpropyl)- o-polyethylene oxide urethane 48 3-(N-styrylmethyl-2- RiceSil 500 3-(N-styrylmethyl-2- 185.33 4.16% 40.42 aminoethylamino)- 100 aminoethylamino)- propyltrimethoxy- propyltrimethoxy-silane silane hydrochloride hydrochloride 49 3- RiceSil 25 3-(Trimethoxysilyl- 3.88 1.90% 24.60 (Trimethoxysilylpropyl) 100 propyl) isothiouronium isothiouronium chloride chloride 50 N-(2-Aminoethyl)-3- RiceSil 500 N-(2-Aminoethyl)-3- 43.42 23.47 2.53% 16.35 amino- 100 aminopropyltrimethoxy- propyltrimethoxysilane, silane plus Glycidyltrimethyl- ammonium chloride 51 3- RiceSil 200 3- 21.53 1.00% 6.75 (Trihydroxysilylpropyl- 100 (Trihydroxysilylpropyl- methylphosphonate) methylphosphonate) sodium salt sodium salt 52 N- RiceSil 500 N- 9.33 0.68% 0.90 Octadecyldimethyl(3- 100 Octadecyldimethyl(3- Trimethoxysilylpropyl) Trimethoxysilylpropyl) ammonium ammonium chloride chloride 53 N-(Trimethoxysilyl- RiceSil 500 N- 5.80 1.50% 12.53 propyl) 100 (Trimethoxysilylpropyl) ethylenediamine, ethylenediamine, triacetic acid, triacetic acid, trisodium trisodium salt salt

TABLE 5B Silane Added Sample Silica Treatment Purity Silane g Ligand # g Silane Type condition % g % C Density 54 500 trimethoxysilylpropyl-ethylenediamine, Toluene, Reflux, 45.0 72.46 1.86 4.79 triacetic acid, trisodium salt H₂O 55 500 N-(triethoxysilylpropyl)-O- Toluene, Reflux, 95.0 59.68 2.34 4.39 polyethylene oxide urethane H₂O 56 500 Bis-(2-hydroxyethyl)-3- Toluene, Reflux, 57.6 22.55 0.93 1.26 aminopropyltriethoxysilane H₂O 57 500 ((chloromethyl)phenylethyl)trimethoxy Toluene, Reflux, 90.0 8.70 1.05 1.55 silane H₂O 58 500 N-(3-triethoxysilylpropyl)-gluconamide Toluene, Reflux, 50.0 31.64 1.12 2.1 H₂O 59 500 3-mercaptopropyltriethoxysilane Toluene, Reflux, 95.0 9.95 0.81 3.59 H₂O 60 500 N-(triethoxysilylpropyl)-4- Toluene, Reflux, 100.0 12.16 1.16 0.21 hydroxybutyramide H₂O 61 500 3-(triethoxysilyl)propylsuccinic Toluene, Reflux, 95.0 12.73 0.76 1.46 anhydride H₂O 62 500 Tris(3- Toluene, Reflux, 95.0 34.18 1.28 2.13 trimethoxysilylpropyl)isocyanurate H₂O 63 500 2-Hydroxy-4-(3-triethoxysilylpropoxy)- Toluene, Reflux, 95.0 23.23 1.61 1.74 diphenylketone H₂O 64 500 Ureidopropyltrimethoxysilane Toluene, Reflux, 100.0 11.72 0.86 2.03 H₂O 65 500 3-isocyanatopropyltriethoxysilane Toluene, Reflux, 95.0 6.90 0.81 5.31 H₂O 66 500 N-(3-trimethoxysilylpropyl)pyrrole Toluene, Reflux, 100.0 6.08 0.87 3.26 H₂O 67 500 Bis[(3-methyldimethoxysilyl)propyl]- Toluene, Reflux, 100.0 18.92 1.72 1.4 polypropylene oxide H₂O

TABLE 5C Ricesil Ligand 100 Silane Silane % C Density Sample Treatment Weight Purity Weight Robertson Calculated # Silane Type condition gram % gram Microlit umol/m2 68 trimethoxysilylpropyl- Toluene, 500 45% 24.20 1.08 1.27 ethylenediamine, triacetic acid, Reflux, H₂O trisodium salt 69 N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 14.60 0.79 1.28 trimethylammonium chloride Reflux, H₂O 70 2-(4-chlorosulfonylphenyl)- Toluene, 500 50% 24.20 1.28 2.89 ethyltrichlorosilane Reflux, H₂O 71 3-(N-styrylmethyl-2- Toluene, 500 32% 46.30 1.65 4.69 aminoethylamino)- Reflux, H₂O propyltrimethoxysilane hydrochloride 72 triethoxysilylpropylethyl-carbamate Toluene, 500 100% 12.35 1.01 1.60 Reflux, H₂O 73 N-(triethoxysilylpropyl)-O- Toluene, 500 95% 19.94 1.09 1.01 polyethylene oxide urethane Reflux, H₂O 74 3- Toluene, 500 42% 22.45 0.83 2.82 trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodium salt 75 Bis-(2-hydroxyethyl)-3- Toluene, 500 58% 22.55 0.93 1.26 aminopropyltriethoxysilane Reflux, H₂O 76 N-(3-triethoxysilylpropyl)-4,5- Toluene, 500 96% 12.06 1 1.57 dihydroimidazole Reflux, H₂O 77 ((chloromethyl)phenylethyl)trimethoxysilane Toluene, 500 90% 8.70 1.05 1.55 Reflux, H₂O 78 3-aminopropyltrimethoxysilane, Toluene, 500 81% 8.15 0.99 2.58 then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 5.03 polyethylene oxide urethane 79 3- Toluene, 500 42% 16.87 0.77 2.43 trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodium salt, then N-(triethoxysilylpropyl)-O- 95% 5.02 polyethylene oxide urethane 80 N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 15.30 0.95 2.41 trimethylammonium chloride, then Reflux, H₂O (3- 100% 2.40 glycidoxypropyl)trimethoxysilane 81 3- Toluene, 500 42% 16.90 0.98 3.81 trihydrosilylpropylmethylphosphonate, Reflux, H₂O sodium salt, then Bis-(2-hydroxyethyl)-3- 58% 5.31 aminopropyltriethoxysilane 82 3-(N-styrylmethyl-2- Toluene, 500 32% 34.76 1.72 4.95 aminoethylamino)- Reflux, H₂O propyltrimethoxysilane hydrochloride, then N-(triethoxysilylpropyl)-O- 95% 5.04 polyethylene oxide urethane 83 2-(trimethoxysilylethyl)pyridine Toluene, 500 100% 9.01 1.17 2.89 Reflux, H₂O 84 N-(3-triethoxysilylpropyl)- Toluene, 500 50% 31.64 1.12 2.10 gluconamide Reflux, H₂O 85 2-(trimethoxysilylethyl)pyridine, Toluene, 500 100% 6.74 1.04 2.40 then Reflux, H₂O N-(3-triethoxysilylpropyl)- 50% 7.90 gluconamide 86 3-mercaptopropyltriethoxysilane Toluene, 500 95% 9.95 0.81 3.59 Reflux, H₂O 87 N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 15.30 0.98 2.54 trimethylammonium chloride, then Reflux, H₂O N-(3-triethoxysilylpropyl)- 50% 7.95 gluconamide 88 N-(triethoxysilylpropyl)-4- Toluene, 500 100% 12.16 1.16 0.21 hydroxybutyramide Reflux, H₂O 89 3-(triethoxysilyl)propylsuccinic Toluene, 500 95% 12.73 0.78 1.42 anhydride Reflux, H₂O 90 Trimethoxysilylpropyl Toluene, 50 50% 1.00 1.04 1.43 polyethyleneimine Reflux, H₂O 91 Tris(3- Toluene, 500 95% 34.18 1.28 1.60 trimethoxysilylpropyl)isocyanurate Reflux, H₂O 92 2-Hydroxy-4-(3- Toluene, 500 95% 23.23 1.61 1.74 triethoxysilylpropoxy)- Reflux, H₂O diphenylketone 93 Ureidopropyltrimethoxysilane Toluene, 500 100% 11.72 0.86 2.03 Reflux, H₂O 94 O-(propargyloxy)-N- Toluene, 500 90% 17.77 1.04 1.84 (triethoxysilylpropyl)urethane Reflux, H₂O 95 3- Toluene, 500 42% 9.33 0.21 0.24 (trimethoxysilyl)propyloctadecyldi Reflux, H₂O methylammonium chloride 96 N-1-phenylethyl-N′- Toluene, 500 100% 9.70 1.02 2.43 triethoxysilylpropylurea Reflux, H₂O 97 3-isocyanatopropyltriethoxysilane Toluene, 500 95% 6.90 0.81 5.31 Reflux, H₂O 98 2-(3,4- Toluene, 500 97% 6.90 0.98 3.22 epoxycyclohexyl)ethyltrimethoxysilane Reflux, H₂O 99 N-(3-trimethoxysilylpropyl)pyrrole Toluene, 500 100% 6.08 0.87 3.26 Reflux, H₂O 100 Bis[(3- Toluene, 500 100% 18.96 1.07 1.40 methyldimethoxysilyl)propyl]- Reflux, H₂O polypropylene oxide 101 N-trimethoxysilylpropyl-N,N,N—Cl, Toluene, 500 50% 10.34 0.84 3.67 trimethylammonium chloride, then Reflux, H₂O 2-Hydroxy-4-(3- 95% 3.12 triethoxysilylpropoxy)- diphenylketone 102 Trimethoxysilylpropylisothiouronium Toluene, 500 43% 15.55 0.71 0.44 chloride Reflux, H₂O 103 (3- Toluene, 500 100% 6.23 0.74 0.35 glycidoxypropyl)trimethoxysilane Reflux, H₂O 104 3-mercaptopropyltriethoxysilane, Toluene, 500 95% 4.98 0.79 1.14 then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 3.38 polyethylene oxide urethane 105 3-(triethoxysilyl)propylsuccinic Toluene, 500 95% 6.36 0.94 1.05 anhydride, then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 3.40 polyethylene oxide urethane 106 trimethoxysilylpropyl- Toluene, 500 45% 20.35 1.16 1.64 ethylenediamine, triacetic acid, Reflux, H₂O trisodium salt, then N-(triethoxysilylpropyl)-O- 95% 3.45 polyethylene oxide urethane 107 2-(4-chlorosulfonylphenyl)- Toluene, 500 50% 13.40 0.93 0.89 ethyltrichlorosilane, then Reflux, H₂O N-(triethoxysilylpropyl)-O- 95% 3.40 polyethylene oxide urethane 108 2-(4-chlorosulfonylphenyl)- Toluene, 500 50% 15.30 1.01 1.15 ethyltrichlorosilane, then Reflux, H₂O Bis-(2-hydroxyethyl)-3- 58% 5.32 aminopropyltriethoxysilane Ligand densities are corrected for 0.43% C due to residual carbon on the original rice hull ash. Mixed silanes sample ligand density are based on first silane.

Example 6 Surface Treated Rice Hull Ash for Protein Binding and Release Objective

To test the binding and release of protein using surface treated rice hull ash (RHA). The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 6 summarizes the filter media samples and their surface treatments.

TABLE 6 Sample Designation Treatment 6 3-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloride treated RHA 4 3-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloride treated RHA Unground RHA Untreated from Producers FW12 Commercial diatomaceous earth (Eager Picher) HQ50 Commercial quaternary amine ion-exchange resin (PerSeptive BioSystems)

Procedure

-   -   1.2 g of each sample was measured into a 50-mL conical tube.     -   2. 25 mL of 25 mM Tris-HCL, pH 8.4 buffer was added.     -   3. Sample and buffer were mixed by inversion overnight.     -   4. Each of the wetted samples was transferred to a 15 mL conical         tube, then centrifuged at 2500 g for 5 minutes and the         supernatant was decanted. The resulting samples were used for         binding test below.     -   5. Protein test solution description and preparation:         -   Source: Micrococcus luteus particulate free concentrated             broth recovered using the following steps:             -   Fermentation broth was lysed using 200 ppm lysozyme                 (from chicken hen white).             -   Lysed broth was flocculated using a poly-cationic                 polymer and filtered to remove particulates.             -   Particulate broth was concentrated using an ultrafilter                 to dewater (Prep/Scale™ TFF, Millipore).     -   6. The above solution was adjusted with 24 parts of 25 mM         Tris-HCl pH 8.4 buffer.     -   7. 5 mL of protein test solution was added to each tube         containing surface treated rice hull ash.     -   8. The tubes were mixed by inversion for 90 min.     -   9. The mixed tubes were centrifuged at 2500 g for 5 minutes and         the supernatant was decanted. The fraction collected is referred         to as “Flow Through or FT”.     -   10. 5 mL of 25 mM Tris-HCl pH 8.4 buffer was added to each of         the tubes which were allowed to mix by inversion for 45 min.     -   11. The tubes were centrifuged at 2500 g for 5 min and the         supernatant was decanted. The fraction collected is referred to         as “Wash”.     -   12. 5 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 2M         NaCl) was added and mixed for 30 min.     -   13. 0.5 mL of 0.5M NaOH was added to each tube.     -   14. The tubes were mixed by inversion for 90 min.     -   15. The tubes were centrifuged at 2500 g for 5 min and the         supernatant was decanted. The fraction collected is referred to         as “Eluate”.     -   16. All the fractions were analyzed by SDS-PAGE gel         electrophoresis (procedure according to NuPAGE Electrophoresis         System, U.S. Pat. No. 5,578,180, by NOVEX electrophoresis GmbH,         Germany).

Observations FIG. 1A (Binding and Analysis of Unbound Components)

Unbound sample was detected by analysis of the Flow Through and the Eluate represents all or a portion of bound sample released in the elution process.

-   -   FW12 (commercial diatomaceous earth) did not bind any protein         from the feed (lane #3 versus lane #2). The slightly lower         intensity for all the bands is due to the dilution by the         solution used to pre-wet the test sample.     -   Untreated RHA selectively bound a protein band above 6 kd and         below 14.4 kd (lane #4 versus lane #2). The slightly lower         intensity for all the bands is due to the dilution by the         solution used to pre-wet the test sample.     -   HQ50 (commercial quaternary amine ion-exchange resin) bound most         of the proteins from the test solution except below 14.4 kds         (lane #5 versus lane #2)     -   Treated RHA Sample 4 selectively bound near and above 97 kd         region, between 55.4 and 36.5 kd, near 21 kd and 14.4 kd         proteins. Note that the bands below 14.4 kd were not captured,         as in the case with HQ50. The overall protein band intensity         appears lower than the untreated rice hull ash and FW12, which         suggests greater binding by treated RHA.     -   Treated RHA Sample 6 demonstrated similar protein binding         selectivity as sample 4 but appears to have lower binding         capacity. Note that the bands below 14.4 kd were not captured,         as in the case with HQ50. The overall protein band intensity         appears lower than the untreated rice hull ash and FW12.

FIG. 1B (Release and Analysis of Bound Components)

-   -   FW12 eluate contains trace amount of proteins which are most         likely from the physically trapped/carried over liquid (lane         #2).     -   The protein, below 14.4 and above 6 kd bands, bound to untreated         RHA were released (lane #3)     -   All the proteins captured by HQ50 were released (lane #4)     -   Eluate from sample 4 contains protein bands above 116 kd, near         and below 55 Kd and near 6 kd. The above 36.5 kd band appears to         remain bound (lane #5).     -   Eluate from sample 6 contain mostly above 116 kd and near 55 kd         bands. Others that were bound either remain bound or are too low         to be detected by the analysis (lane #6).         Untreated diatomaceous earth did not exhibit protein-binding         capability. The untreated rice hull ash demonstrated some         protein binding capability. The two treated rice hull ashes,         sample 4 and 6, demonstrate protein-binding capability.

Example 7 Surface Treated Rice Hull Ash for Protein Binding and Release Objective

To test the binding and release of protein using additional surface treated rice hull ash. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 7 summarizes the filter media samples and their surface treatments.

TABLE 7 Sample Designation Treatment 7 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 8 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 9 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 10 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 11 N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treated 12 N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treated

Procedure Same as in Example 6. Observations Sample 7 Protein Binding and Release (FIG. 2A)

-   -   Selectively bound all MW bands below 55 kd except near 21.5 kd         (lane 2 versus lane 3).     -   The wash has similar profile compared to flow through.     -   The eluate has a very light band near 55 kd, and not many other         bands. The other bands appear to be tightly bound and were not         eluted under conditions used.

Sample 8 Protein Binding and Release (FIG. 2A)

-   -   Similar observations as above, Sample 7.

Sample 9 Protein Binding and Release (FIG. 2B)

-   -   Almost all the proteins were bound except for the band below 14         kd (lane 1 versus lane 2)     -   No protein bands were detected in the wash fraction.     -   Eluate fraction contained mostly near 55 kd band, other bands         remain bound. This demonstrated selective release for the near         55 kd band resulting in a protein purity >90+% based on band         intensity.

Sample 10 Protein Binding and Release (FIG. 2B)

-   -   Similar observations as the sample 9 above

Sample 11 Protein Binding and Release (FIG. 2C)

-   -   Almost all, except some low MW bands, were bound. This         demonstrated selective protein binding. (lane 1 versus lane 3)     -   No protein bands were detected in the wash fraction.     -   Most of the bands bound were eluted under conditions used (lane         5).     -   Appears to have relatively high binding capacity compared to         other surface treated rice hull ashes.

Sample 12 Protein Binding and Release (FIG. 2C) Similar Observations as the Sample 9 Above CONCLUSIONS

Unique protein binding and release were observed for surface treated rice hull ashes. Selective binding was observed (Sample 7 and Sample 8). Selective release (sample 9 and sample 10) resulted in >90% protein purity fractions.

Example 8 Surface Treated Rice Hull Ash for Protein Binding and Release Objective

To test the binding and release of protein using additional surface treated rice hull ash. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 8 summarizes the filter media samples and their surface treatments.

TABLE 8 Treated Rice Hull Ash Identification Surface Treatment 14 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 13 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 17 3-aminopropyltrimethoxysilane treated 18 3-aminopropyltrimethoxysilane treated 19 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated 20 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated surface

Procedure

-   1.2 g of each surface treated rice hull ash was weighed into a 50 mL     conical tube and 40 mL equilibration buffer (25 mM Tris-HCl pH 8.4)     was added. The tubes were mixed by inversion for 30 min. -   2. The tubes were centrifuged at 2500×g for 5 minutes and the     supernatant was decanted. -   3. Protein test solution source: Micrococcus luteus particulate free     concentrated broth was prepared as in Example I followed by partial     digestion using 10 ppm protease. -   4. The above solution was adjusted with 24 parts of 25 mM Tris-HCl     pH 8.4 buffer. -   5. 20 mL of protein test solution was added to each prepared surface     treated rice hull ash. -   6. The samples were mixed by inversion for 30 min. -   7. The samples were centrifuged at 2500×g for 5 minutes and the     supernatant was decanted. The fraction collected is referred to as     “Flow Through or FT” -   8. 20 mL of 25 mM Tris-HCl pH 8.4 buffer was added to the samples     which were allowed to mix by inversion for 15 min. -   9. The samples were centrifuged at 2500×g for 5 min and the     supernatant was decanted. The fraction collected is referred to as     “Wash”. -   10. 20 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 1M NaCl)     was added to each sample and the samples were mixed by inversion for     30 min. -   11. The samples were centrifuged at 2500×g for 5 min and the     supernatant was decanted. The fraction collected is referred to as     “Eluate #1” -   12. Steps 9 and 10 were repeated using 10 mL of the same elution     buffer+50 mM NaOH. The fraction collected is referred to as “Eluate     #2”. -   13. All of the fractions were analyzed by SDS-PAGE gel     electrophoresis.

Observations Sample 14 Protein Binding and Release (FIG. 3A)

-   -   The flow through fraction has relatively low protein band         intensity, which indicates sample 14 has relatively good binding         capacity (lane #6 versus lane #1)     -   The band below 14.4 kd remains in the flow through, which         indicates selective binding.     -   The bound proteins were partially eluted by 1M NaCl.     -   Addition of NaOH to the 1M NaCl containing buffer further eluted         the bound proteins.

Sample 13 Protein Binding and Release (FIG. 3B)

-   -   All the feed proteins were bound (lane #3 versus lane #2)     -   Only a small amount of bound protein was eluted at 1M NaCl (lane         #5), which suggests that the binding may not be ion exchange.     -   Addition of caustic to the 1M NaCl elution buffer successfully         eluted bound protein.     -   The behavior was similar to sample 14.

Sample 17 Protein Binding and Release (FIG. 3C)

-   -   Relatively good binding as shown in lane #7 flow through         fraction.     -   Selectively did not bind the below 14 kd protein.     -   Required high NaCl/NaOH for elution.

Sample 18 Protein Binding and Release (FIG. 3C)

-   -   Relatively good binding as shown in lane #1 flow through         fraction.     -   Selectively did not bind the below 14 kd protein.     -   Required high NaCl/NaOH for elution.     -   The results are similar to those of sample 17.

Sample 19 Protein Binding and Release (FIG. 3D)

-   -   Relatively good binding, as shown in the flow through fraction         on lane #7 having low protein bands.     -   Selectively did not bind the below 14 kd band.     -   Most of the bound proteins were eluted at 1M NaCl.     -   The addition of NaOH to 1M NaCl containing buffer further eluted         the near 55 kd bands.

Sample 20 Protein Binding and Release (FIG. 3E)

-   -   Relatively good binding as indicated by the low protein bands in         the flow through fraction (lane #3).     -   Some leakage during wash (lane #4).     -   Selectively did not bind the below 14 kd band (lane #3).     -   The bound proteins were eluted mostly at 1M NaCl (lane #5).     -   The results are similar to those of sample 19.

CONCLUSIONS

For the above surface treated rice hull ash samples tested, three general binding/release behaviors were observed when the samples were tested under conditions suitable for binding based on anion exchange and release by high salt and/or high pH: Relatively good binding, elute with NaCl/NaOH: Sample 14 (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated) Sample 13 (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated) Sample 17 (3-aminopropyltrimethoxysilane treated) Sample 18 (3-aminopropyltrimethoxysilane treated) Relatively good binding, elute with NaCl: Sample 19 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated) Sample 20 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated) The binding/release test was designed to test for anion exchange behavior. The observations are consistent with the RHA surface modifications. The responses of sample 14 and sample 13 are consistent with a combination of ion exchange and hydrophobic characteristics. Sample 17 and sample 18 also demonstrated a mixture of behaviors. Sample 19 and sample 20 have typical characteristics similar to anion-exchange behavior in terms of both binding and release.

Example 9 Surface Treated Rice Hull Ash for Protein Binding and Release (Cation Exchange) Objective

To test the binding and release of protein using surface treated rice hull ash. The protein solution is particulate free, derived from Aspergillus niger fermentation. Table 9 summarizes the samples designation and their surface treatments.

TABLE 9 Treated Rice Hull Ash Identification Surface 41 1^(st) step 3-glycidoxypropyltrimethoxysilane and 2^(nd) step Na₂S₂O₅ treatment Unground Untreated RHA from Producers Porous HS50 Commercial - SH cation exchange resin (PerSeptive BioSystems, Farmington, MA)

Procedure

-   1.2 g of each surface treated rice hull ash were placed into a 50 mL     conical tube and 40 mL equilibration buffer (100 mM Sodium Acetate,     pH 4.0) was added. The tubes were mixed by inversion for 30 min. -   2. The tubes were centrifuged at 2500×g for 5 minutes and the     supernatant was decanted. -   3. Protein test solution description and preparation:     -   a. Source: Aspergillus niger particulate free concentrated broth         recovered using the following steps:         -   i. The fermentation broth was filtered to remove cell.         -   ii. Ultrafilter (dewater (Prep/Scale™ TFF, Millipore) cell             free broth to dewater.     -   b. The above solution was adjusted with 14 parts of 100 mM         Sodium Acetate, pH 4.0 buffer. -   4. 20 mL of protein test solution was added to each prepared surface     treated rice hull ash. -   5. The samples were mixed by inversion for 70 min. -   6. The samples were centrifuged at 2500×g for 5 minutes and the     supernatant was decanted. The fraction collected is referred to as     “Flow Through or FT”. -   7. 20 mL of 100 mM Sodium Acetate pH 4.0 buffer was added to each     sample, and the samples were allowed to mix by inversion for 15 min. -   8. The samples were centrifuged at 2500×g for 5 min and the     supernatant was decanted. The fraction collected is referred to as     “Wash #1”. -   9. Steps 7 & 8 were repeated and the fraction collected is referred     to as “Wash #2” -   10. 20 mL elution buffer (100 mM Sodium Acetate pH 4.0 buffer     containing 1M NaCl) was added and the samples were mixed by     inversion for 60 min. -   11. The samples were centrifuged at 2500 g for 5 min and the     supernatant was decanted. The fraction collected is referred to as     “Eluate #1” -   12. Repeated step 9 and 10 using 10 mL of the same elution buffer+50     mM NaOH. The fraction collected is referred to as “Eluate #2”. -   13. All the fractions were analyzed by SDS-PAGE gel electrophoresis.

Observations Sample 41 Protein Binding and Release (FIG. 4A)

-   -   Selectively binds near 97 kd and below 31 kd bands.     -   There were relatively low to no protein bands detected in the         “Washes #1 and #2”, respectively (see lane #3 and lane #4,         respectively), which implies that the binding was         specific/strong.     -   The bound proteins were eluted in 1M NaCl containing buffer.

Untreated RHA Protein Binding and Release (FIG. 3A)

-   -   The near 97 kd and below 31 kd bands were not present in the         flow through. However, no proteins were eluted in either Eluate         #1 or Eluate #2.

Porous HS50: Protein Binding and Release (FIG. 4B)

-   -   Selectively binds near 97 kd and below 31 kd bands.     -   There were relatively low to no protein bands detected in the         “Washes #1 and #2”, respectively (see lane #3 and lane #4,         respectively), which suggests that the binding was         specific/strong.     -   The bound proteins were eluted in 1M NaCl containing buffer.

CONCLUSION

The surface treated rice hull ash sample 41 has very similar binding and release characteristics to the positive control.

Example 10 Surface Treated Silica Filter Media for Protein Binding and Release (Ion Exchange) Objective

To test the binding and release of protein using surface treated silica filter media. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 10 summarizes the samples designation and their surface treatments.

TABLE 10 Sample Identification Description Sample 42 1^(st) step 3-aminopropyltrimethoxysilane and 2^(nd) step Glycidyltrimethylammonium chloride treatment Sample 40 1^(st) step N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and 2^(nd) step Glycidyltrimethylammonium chloride treatment Sample 34 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celite 545 Sample 29 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celpure P1000 (commercial diatomaceous earth) AgriSilicas Untreated RHA Celite 512 Untreated commercial diatomaceous earth (World Minerals)

Procedure

Same as in Example 8 for all samples except sample 29, sample 30 and CelPure P100, which have the following variations: The protein test solution was diluted by 10× (versus 25×). Steps 4 and 5 were repeated and the wash fraction collected is referred to as “Wash #2”.

Observations

Under the test conditions used, the amount of protein test solution was in excess. As a result, all the flow through fractions had similar protein band patterns compared to the feed test solution. No attempt was made to qualitatively describe the protein binding capability of each silica filter media sample tested. The following observations are based on the eluate fractions only.

Sample 42 (FIG. 5A)

Most of the bound proteins were eluted at 1M NaCl (lane #5).

Sample 40 (FIG. 5B)

Most of the bound proteins were eluted at 1M NaCl (lane #5).

Sample 34 (FIG. 5C)

No significant amount of protein was eluted at 1M NaCl. Small amount of proteins were eluted subsequently using high pH.

Sample 29 (FIG. 5D)

Both eluate fractions contain proteins, and the compositions seem similar in these fractions (lane #5 for 1M NaCl eluate and lane #6 for high pH eluate).

Untreated AgriSilica RHA (FIG. 5B)

The eluted fractions contain proteins, especially at MW lower than 14.4 kd.

Celite 512 (FIG. 5C)

The fraction eluted at 1M contains proteins near 97 kd, near and below 55 kd and especially between 14.4 kd and 6 kd (lane #10).

CONCLUSION

Samples 40 and 42 (surface treated rice hull ash) and samples 29 and 34 (surface treated diatomaceous) demonstrate protein-binding capability over the corresponding untreated counterparts.

Example 11 Surface Treated Rice Hull Ash for Dynamic Protein Binding and Release (Ion Exchange) Objective

To test the dynamic binding and release of protein using surface treated rice hull ash sample 9. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation.

Procedure

-   1. 6 g of sample 9 was placed into a 50 mL conical tube. -   2. 50 mL equilibration buffer (25 mM Tris-HCl pH 8.4) was added and     the sample was mixed by inversion for 30 min. -   3. The samples was centrifuged at 2500 g for 5 minutes and the     supernatant was decanted. -   4. 30 mL of equilibration buffer was added and the sample was mixed     well by inversions. -   5. The sample was poured into a gravity flow column. -   6. The surface-treated rice hull ash was allowed to settle and pack     to a 10 mL volume. -   7. The pre-filter was placed onto the packed bed. -   8. 20 ml of equilibration buffer was added. -   9. 25 mL of protein test solution was added (prepared the same way     as in Example 6) -   10. Flow through fractions were collected in 15 mL conical tubes. -   11. 30 mL of equilibration buffer was added, and the “wash” was     collected in 15 ml conical tubes. -   12. The following steps were used sequentially for election and     collection of multiple elutes as shown in Table 11:     -   a. 0.2M NaCl in equilibration buffer was added.     -   b. 2M NaCl in equilibration buffer was added.     -   c. 0.1M NaOH was added. -   13. All the fractions were analyzed by SDS-PAGE gel electrophoresis.

Observations (FIG. 6)

-   -   The amount of solution loaded was higher than the capacity,         hence significant breakthrough in the FT fractions (lanes 3, 4         and 5)     -   The surface treated rice hull ash, sample 9, had good flow         property. All the steps performed above were easily accomplished         by gravity flow.     -   FIG. 6 shows that at the at 10 mL load, the feed solution         appears to breakthrough the 10 mL packed sample 9.     -   Under the binding conditions tested, sample 9 appears to         selectively bind the near 96 kd, near 55 kd, the two bands below         the 55 kd, bands near and between the 14.4 kd and 6 kd.     -   The following were observed with the three elution steps:         -   Three bands (near 97 kd, near 55 kd, and below 14.4 kd) were             eluted at 0.2M.         -   At 2M NaCl, near 97 kd and near 55 kd bands were eluted.         -   Under 0.1M NaOH, near 55 kd, below 31 kd bands and near 14.4             kd proteins were eluted.             Table 11 shows a summary of fractions collected for the             binding test.

TABLE 11 Volume Fraction (mL) Feed 25 mL loaded FT#1 10.5 mL FT#2 (after 10 mL feed was loaded) 10 mL FT#3 (after 14.5 mL was loaded) 4.5 mL FT#4 (after 25 mL feed was loaded) 10 mL Wash 30 mL Eluate #1 (0.2M NaCl) 10 mL Eluate #2 (2M NaCl) 10 mL Eluate #3 (1st 0.1M NaOH fraction, very dark) 4.5 mL Eluate #4 (2^(nd) 0.1M NaOH) 3.5 mL

CONCLUSIONS

This example demonstrates that surface-treated rice hull ash can be used in a packed bed chromatography mode for protein binding and release and as a filter aid with gravity flow alone. The binding and release characteristics are similar to those of batch mode. The example also illustrates that selective elution can be achieved by using different elution buffers.

Example 12 Surface-Treated Rice Hull Ash for Simultaneous Particulate Capture and Soluble Capture/Release Objective

To test the characteristics of surface-treated rice hull ash for simultaneous particulate filtration, and protein binding and release. The surface treated rice hull ash was designated sample 19, which was demonstrated to have anion exchange characteristics (see Example 8). The untreated rice hull ash was also tested in parallel.

Buffers Equilibration Buffer: 25 mM Tris-HCl, pH 8.4. Elution Buffer: 25 mM Tris-HCl, 1M NaCl, pH 8.4; 1M NaOH; 1M HCl Test Solution

Flocculated Micrococcus luteus fermentation broth referred to as “feed” was prepared according to the following:

-   -   After harvest, the broth was lyzed using 100 ppm lysozyme         (chicken egg white).     -   The lysed broth was flocculated using poly-cationic polymer.     -   The flocculated sample was diluted with 1 part equilibration         buffer before testing.

Procedure

-   1. Surface-treated rice hull ash preparation:     -   5 g of untreated RHA was placed into each of the two 50 mL         conical tubes.     -   40 mL equilibration buffer was added and the tubes were mixed by         inversion for 30 min. -   2. The tubes were centrifuged at 2500×g for 5 minutes and decanted     in step #1 for the untreated rice hull ash. -   3. 50 mL of the prepared test solution “feed” was added to each     prepared rice hull ash. -   4. The tubes were mixed by inversion for 30 min at room temperature. -   5. A 1 mL small sample was centrifuged using a bench top centrifuge     (4 min) and the supernatant was collected (referred to as “Bench     FT”). -   6. 0.45 μm 250 mL-Nalgen unit was prepared for filtration:     -   The unit was connected to a house vacuum outlet.     -   The other prepared rice hull ash was suspended in 50 mL of         equilibration buffer.     -   The suspension was poured into the filter unit, and (house)         vacuum was applied to form a pre-coat (cake).     -   The filtrate reservoir was emptied.     -   The reservoir was reconnected for the filtration test. -   7. The protein solution with rice hull ash ad-mix from step 4 was     poured into the prepared filtration unit and vacuum was reapplied to     start filtration. The collected filtrate sample is referred to as     “FT Filtrate”. -   8. The vacuum was discontinued and 50 mL of Equilibration Buffer was     added and mixed by stirring. The vacuum was reapplied to start     filtration. The filtrate sample was collected and referred to as     “Wash”. -   9. Step 8 was repeated with 50 mL of Elution Buffer and mixed for 15     min before vacuum was reapplied to start filtration. The filtrate     sample was collected and is referred to as “Eluate”. -   10. All the fractions were analyzed by 4-12% Tris-Bis SDS-PAGE gel     electrophoresis with MES running buffer (see separate Excel file for     procedure). -   11. Steps 1-10 were repeated with the untreated rice hull ash.

Observations/Comments

-   1. The surface-treated rice hull ash, sample 19, appears to have     slightly thinner cake thickness than the untreated rice hull ash. -   2. All the fractions collected (FT filtrate, wash and eluate) from     both rice hull ashes were clear, free of particulate. -   3. The surface-treated rice hull ash, sample 19, has a particulate     filtration rate comparable to the filtration rate of the untreated     rice hull ash:     -   Sample 19: 12.8 mL/min     -   Untreated RHA: 14.0 mL/min -   4. Sample 19 demonstrates good capture and release over untreated     RHA:     -   Untreated RHA (FIG. 7A)         -   The “FT filtrate” (lane #4) has very similar profile as the             feed (lane #2). All the bands are slightly lighter than the             feed, which is an artifact of dilution from the buffer used             to condition the rice hull ash.         -   The protein solution physically trapped within the rice hull             ash was displaced and this is represented by the “wash”             fraction (contains very faint protein bands, see lane #5)         -   There was only trace amount of protein in the Eluate (lane             #6). -   5. Sample 19 (FIG. 7B)     -   Demonstrates good binding and recovery of the bound protein.     -   The “FT filtrate” fraction has very low to no protein (lane #4).         The “Bench FT” supernatant (lane #3) has slight protein bands         when compare to the “FT Filtrate”, which indicates that proteins         were captured as they passed through the cake.     -   The wash has low to no protein bands (lane #5).     -   The Eluate has similar band patterns but slightly less intense         than the feed (lane #6).

CONCLUSION

The surface-treated rice hull ash simultaneously captured soluble proteins of interest by ion exchange and separated particulates from the feed protein solution. The captured proteins can be subsequently extracted from the surface treated rice hull ash by elution with a high-salt buffer. The results demonstrate that surface-treated rice hull ash can be used to separate a particulate-containing protein solution into three streams: particulates trapped in surface-treated rice hull ash pre-coat and body feed, non-protein components bound to surface treated rice hull ash, and protein components bound to and eluted off the surface treated rice hull ash.

Example 13 Surface Treated Rice Hull Ash for Simultaneous Particulate Capture and Soluble Capture/Release Objective

To repeat Example 12 using a Aspergillus niger broth using the same surface-treated rice hull ash (sample 19) and untreated rice hull ash.

Test Solution

Aspergillus niger fermentation was diluted with 4 parts of DI water and pH was adjusted to 8.06 using NaOH.

Procedure

Same as in the Example 12. Test solution volume was 100 mL.

Observations/Comments

The surface treated rice hull ash, sample 19, has a comparable particulate filtration rate to the untreated rice hull ash. All the fractions collected (FT filtrate, wash and eluate) from both rice hull ashes were clear, free of particulate. Under the conditions tested, the amount of test solution used was in excess of the binding capacity. As a result, the flow through fractions (both “bench FT” and “Filtrate FT”) for both sample 19 and untreated RHA were not significantly different from the feed solution. See FIG. 8, lanes #2, 3 and 4 versus lane #1 for untreated RHA and lanes #7, 8 and 9 versus lane #1 for sample 19. The following observations confirmed that sample 19 has protein-binding capability over the untreated RHA (see FIG. 8): Untreated RHA Wash (lane #5) contains more protein than the sample 19 (lane #10). The eluted fraction from sample 19 (lane #11) shows higher protein band intensity than the eluted fraction from untreated RHA (lane #6).

CONCLUSION

This example demonstrates that the surface-treated rice hull ash simultaneously captured soluble proteins of interest by ion exchange and separated particulates from the Aspergillus niger derived feed protein solution. The captured proteins can be subsequently extracted from the surface treated rice hull ash by elution with high salt buffer. The results demonstrate that surface-treated rice hull ash can be used to separate a particulate containing protein solution into three streams: particulates trapped in surface treated rice hull ash pre-coat and body feed, non-protein components bound to surface treated rice hull ash, and protein components bound to and eluted off the surface treated rice hull ash.

Example 14 Protein Binding Test Materials MilliQ H₂O

Protein solution (filtered catalase DFC) 50 mL Oak Ridge tubes Sorval RC 5B Plus centrifuge with Sorval SA600 rotor BCA Protein assay kit (Pierce)

Compat-Able Protein Assay Preparation Reagent Set (Pierce)

Pre-Diluted Protein Assay Standards, bovine serum albumin fraction V set (Pierce) 5 μm syringe filter (Sartoris, Minisart, #17594)

Procedures

-   1. 1 g of each silane treated rice hull ash Sample #54-67 was added     into a 50 mL Oak Ridge tube. -   2. 20 mL MilliQ H₂O was added to each tube. -   3. The contents of each tube was mixed by turning end-over-end at 8     rpm for 10 minutes at room temperature. -   4. Each tube was centrifuged at 16,000 rpm, 15° C. for 15 minutes. -   5. The supernatant was carefully removed using plastic transfer     pipettes. -   6. 1 part protein solution was diluted with 24 parts MilliQ H₂O     (Feed). -   7. 10 mL Feed was added to each tube (gave 10% w/v solid). -   8. Each tube was incubated at room temperature for 2 hours, turning     end-over-end at 8 rpm. -   9. Each tube was centrifuged at 16,000 rpm, 15° C. for 30 minutes. -   10. Each supernatant was filtered through a 0.45 μm syringe filters. -   11. The protein concentrations of Feed and filtered supernatants     (Step 10) were measured by BCA assay using microtiterplate protocol.     The results are shown as Table 12.

TABLE 12 Protein concentration ug/ml Sample # Silane Type Original 160.66 Untreated RHA 98.75 54 trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium 120.09 salt 55 N-(triethoxysilylpropyl)-O-polyethylene oxide urethane 136.10 56 Bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane 112.02 57 ((chloromethyl)phenylethyl)trimethoxysilane 103.21 58 N-(3-triethoxysilylpropyl)-gluconamide 81.02 59 3-mercaptopropyltriethoxysilane 73.43 60 N-(triethoxysilylpropyl)-4-hydroxybutyramide 98.83 61 3-(triethoxysilyl)propylsuccinic anhydride 73.49 62 Tris(3-trimethoxysilylpropyl)isocyanurate 81.41 63 2-Hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone 49.57 64 Ureidopropyltrimethoxysilane 100.04 65 3-isocyanatopropyltriethoxysilane 76.12 66 N-(3-trimethoxysilylpropyl)pyrrole 59.32 67 Bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide 110.24

Results

The protein concentrations of the Feed materials were decreased after the Feed materials were mixed with sample numbers 54-67, centrifuged, and filtered. The results indicate that silane-treated silica sample numbers 54-67 and untreated RHA all bound proteins from the Feed protein solution.

Example 15 Test of Antimicrobial Activity (Bacillus subtilis)

Microorganism tested: Bacillus subtilis Filter media tested: filter media samples 43, 44, 4 and FW12 (untreated diatomaceous earth)

Protocol:

-   -   Bacillius subtilis fermentation broth was diluted in sterile PBS         to ˜10⁴ CFU/mL (1 OD≈5*10⁸ CFU/mL was used to estimate CFU/mL in         fermentation broth)     -   Use 0.5 g filter media/5 mL liquid (10% solid)

-   1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted     broth sample were plated on LA plates to determine actual CFU/mL     used. Plates were incubated over night at 34° C.

-   2. Filter media and diluted bacterial sample (or PBS control) were     mixed in a sterile 125 mL baffled flask for 2½ hours at 30° C., 200     rpm.

-   3. Liquid part of the treated samples (2) were plated on LA plates     (5 plates for each sample, one plate for control) and incubated     overnight at 34° C.

-   4. The plates were counted for bacteria.

Results:

The results are summarized in Table 13. By mixing the bacteria with filter media samples 4 and 44, the CFUs were reduced, which indicates that filter media samples 4 and 44 had anti-microbial activity and killed the bacteria by contacting.

TABLE 13 Sample CFU/mL Diluted broth - start 6.53 * 10³ ± 2.47 * 10³ Sample 43 + bacteria - mixing 1.04 * 10⁴ ± 1.50 * 10³ Sample 44 + bacteria - mixing 1.30 * 10² ± 3.00 * 10¹ Sample 4 + bacteria - mixing TFTC FW12 + bacteria - mixing 5.90 * 10⁴ ± 8.00 * 10³ Diluted broth sample - mixing 1.05 * 10³ ± 5.00 * 10¹

Notes and Abbreviations:

-   -   PBS: Phosphate buffered saline (prevents cells from lysing due         to osmotic chock)     -   CFU: colony forming units (a measure of viable cells)     -   TFTC: Too Few To Count     -   The CFU/mL are reported as: Average±Difference (number of         plates) [the difference is between the average and the         observations farthest from the average].     -   Only plates with between 20-300 colonies were counted.

Example 16 Test of Antimicrobial Activity (Bacillus subtilis)

Microorganism tested: Bacillus subtilis Filter Media tested: filter media samples 1, 4, 6, 44, and 45.

Protocol:

Bacillus subtilis fermentation broth was diluted in sterile PBS to ˜10⁴ CFU/mL.

0.5 g filter media/5 mL liquid (10% solid) was used.

-   1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted     broth sample were plated on LA plates to determine actual CFU/mL     used. Plates were incubated over night at 34° C. -   2. Filter media and diluted bacterial sample (15 mL liquid) were     mixed in a sterile 250 mL baffled flask. 2 flasks were used for each     filter media.     -   (A flask with PBS instead of bacterial sample was included for         the following filter media: Samples 1, 6 and 45) -   3. The above was mixed for 2 hours at 30° C., 250 rpm. -   4. Treated samples (the liquid part) were plated on LA plates (4 or     5 plates for each sample). Plates were incubated overnight at 34° C. -   5. The plates were counted for bacteria.

Results:

The results are summarized in Table 14. By mixing the bacteria with filter media samples 1, 4, 6, 44, and 45, the CFUs were significantly reduced.

TABLE 14 Sample CFU/mL Diluted broth - start 3.45 * 10⁴ ± 4.50 * 10³ Diluted broth - mixing 1.72 * 10⁴ ± 1.55 * 10³ Sample 1 A TFTC B TFTC Sample 4 A TFTC B TFTC Sample 6 A TFTC B 1.00 * 10² ± 0.00 * 10⁰ Sample 44 A 3.10 * 10² ± 9.00 * 10¹ B 6.00 * 10² Sample 45 A TFTC B TFTC

Example 17 Test of Antimicrobial Activity and Filtration (Lactobacillus brevis)

Microorganism tested: Lactobacillus brevis Filter media tested: Samples 4, 43, 45 & FW12. Used 0.5 g filter media/5 mL culture (10% solid).

Protocol:

-   1. A Lactobacillus brevis overnight culture was diluted to ˜10⁵     CFU/mL (based on 1 OD₆₀₀≈2.7*10₈ CFU/mL) in two steps—the first     dilution was made in sterile Lactobacillus MRS broth, the second in     sterile PBS. -   2. Serial dilutions (in 0.9% w/v NaCl) of the culture were made     (second dilution). -   3. Diluted samples were plated on Lactobacillus MRS broth plates, to     determine actual starting CFU/mL. -   4. Filter media and diluted bacterial sample (10 mL liquid) were     mixed in a sterile 125 mL baffled flask, sealed with PARAFILM®, for     2 hours 15 minutes at room temperature on an orbit shaker (˜60 rpm). -   5. Serial dilutions (in 0.9% w/v NaCl) were made of treated sample     and plated on Lactobacillus MRS broth plates. -   6. Selected samples/dilutions of samples 4, 43 and 45 were filtered     through a 5 μm filter. -   7. The filtered samples were plated on Lactobacillus brevis broth     plates, and incubated in a candle jar at 30° C. for 2 days. -   8. The plates were counted.

Results:

The results are summarized in Table 15. CFUs were reduced by mixing Samples 4, 43, and 45 with bacteria. CFUs were further reduced by filtering the mixture through a 5 μm filter.

TABLE 15 Sample CFU/mL Lactobacillus brevis culture - start 1.05 * 10⁵ ± 2.50 * 10³ Lactobacillus brevis culture - mixing 1.23 * 10⁵ ± 2.50 * 10³ Sample 4 (mixing) 3.22 * 10⁴ ± 4.77 * 10³ Sample 43 (mixing) 3.43 * 10⁴ ± 5.67 * 10³ Sample 45 (mixing) 5.55 * 10² ± 4.50 * 10¹ FW12 (DE) 8.60 * 10⁴ ± 4.75 * 10³ Filtered Sample 4 TFTC Filtered Sample 43 TFTC Filtered Sample 45 TFTC

Example 18 Test of Antimicrobial Activity (E. coli)

Microorganism tested: E. coli (MG1655) Filter media tested: FW12, samples 43, 1, 4, 6, 44 and 45.

Protocol:

0.5 g Filter Media/5 mL Feed (=10% solid).

-   1. An E. coli culture (not yet in stationary phase) was diluted to     ˜10⁵ CFU/mL (based on 1 OD₆₀₀≈5*10⁸ CFU/mL) in two steps—the first     dilution was made in sterile LB media, the second in sterile PBS     (this was the Feed). -   2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made. -   3. 100 μL of the diluted feed samples were plated on LA plates, to     determine the actual starting CFU/mL. -   4. Filter media and 10 mL feed were mixed in a sterile 125 mL     baffled flask for 2 hours at 25° C., 200 rpm (¾ inch stroke). -   5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made     and 100 μl of each was plated on LA plates, and incubated overnight     at 30° C. -   6. Plates were counted.

Results:

The results are summarized in Table 16.

TABLE 16 Sample CFU/mL MG1655 - start 6.80 * 10⁴ ± 4.00 * 10³ MG1655 - mixing 5.35 * 10⁵ ± 2.50 * 10⁴ diatomaceous earth 2.28 * 10⁵ ± 1.72 * 10⁵ Sample 43 9.05 * 10³ ± 5.50 * 10² Sample 1 1.28 * 10³ ± 2.45 * 10² Sample 4 1.73 * 10⁴ ± 2.03 * 10³ Sample 6 TFTC Sample 44 2.70 * 10³ ± 1.23 * 10² Sample 45 5.20 * 10³ ± 2.00 * 10²

Example 19 Test of Antimicrobial Activity and Filtration (Lactobacillus brevis)

Microorganism tested: Lactobacillus brevis type strain (ATCC#14869) Filter media tested: Samples 43, 4, and 44

Protocol:

0.5 g Filter media/5 mL Feed (=10% solid)

-   1. A Lactobacillus brevis culture was diluted to ˜10⁵ CFU/mL (based     on 1 OD₆₀₀≈2.7*10⁸ CFU/mL) in two steps—the first dilution was made     in sterile Lactobacillus MRS broth, the second in sterile PBS (this     was the Feed). -   2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made. -   3. 100 μL of the diluted samples were plated on Lactobacillus MRS     broth plates, to determine the actual starting CFU/mL. -   4. Filter media and 5 mL Feed were mixed in a sterile 15 mL conical     tube for 2 hours at 25° C., 250 rpm (½ inch stroke). -   5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made     and plated on Lactobacillus MRS broth plates (100 μl each). -   6. All samples were filtered through 5 μm syringe filter. -   7. Serial dilutions (in 0.9% w/v NaCl) of filtered samples were made     and plated on Lactobacillus MRS broth plates. -   8. Plates were counted in a candle jar at 30° C. for 2 days. -   9. Plates were counted.

Results:

The results are summarized in Table 17. CFUs were reduced by mixing Samples 4, 43, and 44 with bacteria. CFUs were further reduced by filtering the mixture through a 5 μm filter.

TABLE 17 Sample CFU/mL CFU/mL (filtered) ATCC#14869 - start 2.83 * 10⁴ ± 4.67 * 10³ ATCC#14869 - 4.00 * 10⁴ ± 2.00 * 10³ 1.27 * 10⁴ ± 5.80 * 10² mixing Sample 43 4.55 * 10³ ± 3.50 * 10² 2.40 * 10³ ± 2.00 * 10² Sample 4 1.95 * 10² ± 5.00 * 10⁰ TFTC Sample 44 8.10 * 10² ± 1.40 * 10² 5.50 * 10¹ ± 5.00 * 10⁰

Example 20 Test of Antimicrobial Activity (Lactobacillus brevis)

Microorganism tested: Lactobacillus brevis Filter media tested: Samples 48, 50, 51, and 52.

Protocol:

-   1. Lactobacillus brevis (gram positive) culture was streaked on MRS     agar and incubated anaerobically at 26° C. until growth was     sufficient. -   2. Working inoculum was prepared by diluting colonies from the MRS     plates into 0.1% peptone, targeting 5×104 cfu/mL. -   3. 0.5 g filter media was added to 10 mL inoculum in a 30 mL glass     tube (5%). -   4. The glass tube was sealed and incubated at room temperature for     30 minutes with mixing (8 inversions/minute). -   5. Serial dilutions of 1:10 were prepared in 0.9% NaCl and plated     with MRS agar, using the pour plate method to enumerate bacterial     population. -   6. Plates were incubated at 26° C., anaerobically (GasPak), until     growth was sufficient to count. -   7. Plates that had 20-200 colonies were counted. The Results are     summarized in Table 18.

Example 21 Test of Antimicrobial Activity (Acetobacter pasteurianus (Gram Negative))

Microorganism tested: Acetobacter pasteurianus (gram negative) Filter media tested: Samples 48, 50, 51, and 52.

Protocol:

-   1. Acetobacter pasteurianus (gram negative) culture was streaked     onto MRS agar and incubated aerobically at 27° C. until growth was     sufficient. -   2. Culture was stocked by adding 1 mL loop of agar plate colonies to     99 mL of MRS broth and incubated at 27° C. -   3. Working inoculum was made by diluting an aliquot of the MRS stock     culture into either phosphate buffered saline (PBS) or 0.1% peptone. -   4. 0.5 g of filter media was added to 10 mL inoculum in a 30 mL     glass tube. -   5. The glass tube was sealed and incubated at room temperature for     30 minutes with mixing (8 inversions/minute). -   6. Serial dilutions of 1:10 were performed in 0.1% peptone and     plated with MRS agar, using the pour plate method to enumerate     bacterial population. -   7. Plates were counted at 27° C., aerobically, until growth was     sufficient to count. -   8. Plates that had 20-200 colonies were counted. The Results are     summarized in Table 18.

Example 22 Test of Antimicrobial Activity (Saccharomyces diastaticus (Yeast))

Microorganism tested: Saccharomyces diastaticus (yeast) Filter media tested: Samples 48, 50, and 51.

Protocol:

-   1. Saccharomyces diastaticus (yeast) culture was streaked onto YM     agar and incubated aerobically at 30° C. until growth was     sufficient. -   2. Working inoculum was prepared by diluting colonies from the YM     plates into phosphate buffered saline (PBS), targeting 3×104 cfu/mL. -   3. 0.5 g to filter media was added to 10 mL inoculum in a 30 mL     glass tube. -   4. The glass tube was sealed and incubated at room temperature for     30 minutes with mixing (8 inversions/minute). -   5. Serial dilutions of 1:10 were performed in 0.9% NaCl and plated     with MRS agar, using the pour plate method to enumerate bacterial     population. -   6. Plates were incubated at 30° C., aerobically, until growth was     sufficient to count. -   7. Plates that had 20-200 colonies were counted. The Results are     summarized in Table 18.

TABLE 18 Lactobacillus Acetobacter Saccharomyces Brevis, grams pasteurinus, distaticus, Sample positive (+) gram negative (−) yeast No. Treatment Silica Type % Reduction % Reduction % Reduction 48 3-(N-styrylmethyl-2- RiceSil 100 100% 18% 41% aminoethylamino)- propyltrimethoxy- silane hydrochloride 51 3-trihydroxysilylpropyl- RiceSil 100 20% 10% 33% methyl phosphonate, sodium salt 50 N-(2-Aminoethyl)-3- RiceSil 100 90% 20% 3% aminopropyltrimethoxy- silane Glycidyltrimethyl- ammonium chloride 52 N- RiceSil 100 100% 90% Octadecyldimethyl(3- Trimethoxysilylpropyl) ammonium chloride

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications could be made without departing from the scope of the invention. 

1. Silane-treated silica filter media prepared by reacting the surface active group of silica filter media with one or more silanes comprising a hydrolyzable moiety selected from the group consisting of aryloxy, amide, methacrylate, carbonyl, urethane, pyrrole, carboxy, cyano, aminoacyl, acylamino, alkyl ester, and aryl ester, wherein the silica filter media are rice hull ash, oat hull ash or diamataceous earth.
 2. The silane-treated silica filter media according to claim 1, wherein said hydrolyzable moiety is selected from the group consisting of aryloxy, amide, methacrylate, carbonyl, urethane, pyrrole, and cyano.
 3. The silane-treated silica filter media according to claim 1, wherein said silane has an additional functional moiety selected from the group consisting of quaternary ammonium, aryl, amino, urea, methacrylate, imidazole, carbonyl, isothiorium, sulfonate and phosphonate.
 4. Silane-treated silica filter media prepared by reacting the surface active group of silica filter media with silanes selected from the group consisting of 3-aminopropyltrimethoxysilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 3-trihydrosilylpropylmethylphosphonate, sodium salt and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and (3-glycidoxypropyl)trimethoxysilane; 3-trihydrosilylpropylmethylphosphonate, sodium salt and bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane; 3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 2-(trimethoxysilylethyl)pyridine and N-(3-triethoxysilylpropyl)-gluconamide; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and N-(3-triethoxysilylpropyl)-gluconamide; N-trimethoxysilylpropyl-N,N,N—Cl, trimethylammonium chloride and 2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone; 3-mercaptopropyltriethoxysilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 3-(triethoxysilyl)propylsuccinic anhydride and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium salt and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane; and 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane and bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane, wherein the silica filter media are rice hull ash, oat hull ash or diamataceous earth.
 5. Silane-treated silica filter media prepared by reacting the surface active group of silica filter media with one or more silanes selected from the group consisting of: 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane, 2-hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone, (chloromethyl)phenylethyl)trimethoxysilane, phenyldimethylethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, 2-(trimethoxysilylethyl)pyridine, N-(3-trimethoxysilylpropyl)pyrrole, trimethoxysilylpropyl polyethyleneimine, N-(3-trimethoxysilylpropyl)pyrrole, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, N-(triethoxysilylpropyl)-O-polyethylene oxide urethane, N-(triethoxysilylpropyl)urea, 3-(trimethoxysilyl)propyl methacrylate, 3-(triethoxysilyl)propylsuccinic anhydride, tris(3-trimethoxysilylpropyl)isocyanurate, bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide, N-(triethoxysilylpropyl)-O-polyethylene oxide urethane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, N-[3-(triethoxysilyl)propyl]imidazole, N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole, 2-(4-chlorosulfonylphenyl)-ethyltrichlorosilane, trimethoxysilylpropylisothiouronium chloride, N-(3-triethoxysilylpropyl)-gluconamide, N-(triethoxysilylpropyl)-4-hydroxybutyramide, N-(triethoxysilylpropyl)-O-polyethylene oxide urethane, O-(propargyloxy)-N-(triethoxysilylpropyl)urethane, 3-(trimethoxysilyl)propyl-ethylenediamine triacetic acid trisodium salt, and 3-(trihydroxysilyl)propylmethylposphonate sodium salt, wherein the silica filter media are rice hull ash, oat hull ash, or diamataceous earth.
 6. The silane-treated silica filter media of claim 5, where said one or more silanes are 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane, phenyldimethylethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, N-(triethoxysilylpropyl)urea, 3-(trimethoxysilyl)propyl methacrylate, or N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole. 